SERINE PROTEASE VARIANTS HAVING AMINO ACID DELETIONS AND SUBSTITUTIONS

VARIANTS DE SERINE-PROTEASES AYANT DES DELETIONS ET DES SUBSTITUTIONS D'ACIDES AMINES

English Abstract

The present invention relates to variants of serine proteases having decreased
immunogenicity relative to their corresponding wild-type proteases. More
particularly, the present invention relates to variants having a modified
amino acid sequence of a wild-type amino acid sequence, wherein the modified
amino acid sequence comprises a deletion and, optionally, a substitution of
one or more specifically identified positions corresponding to subtilisin
BPN'. The invention further relates to mutant genes encoding such variants and
cleaning and personal care compositions comprising such variants.

French Abstract

Cette invention se rapporte à des variants de sérine-protéases ayant une immunogénicité inférieure à celle des protéases du type sauvage correspondantes. Cette invention se rapporte plus particulièrement à des variants ayant une séquence d'acides aminés modifiée par rapport à une séquence d'acides aminés de type sauvage, cette séquence d'acides aminés modifiée comportant une délétion et, éventuellement, une substitution d'une ou de plusieurs positions spécifiquement identifiées correspondant à la subtilisine BPN'. Cette invention se rapporte en outre à des gènes mutants codant ces variants et à des compositions de produits de nettoyage et de produits de soins personnels comprenant ces variants.

Claims

87

What is claimed is:

1. A variant of a serine protease characterized by a modified amino acid
sequence
of a wild-type amino acid sequence, wherein the modified amino acid sequence
comprises a deletion of one or more of positions 70 - 84 corresponding to
subtilisin
BPN', wherein the variant is not a 75 - 83 or .DELTA. 71.
2. A variant according to Claim 1 wherein the serine protease is selected from
the
group consisting of subtilisin BPN', subtilisin Carlsberg, subtilisin DY,
subtilisin 309,
proteinase K, and thermitase.
3. A variant according to Claims 1 or 2 wherein the deletion is of one or more
of
positions 73 - 83 corresponding to subtilisin BPN'.
4. A variant according to any of the preceding claims wherein the deletion is
of
one or more of positions 70 and 75 - 82 corresponding to subtilisin BPN'.
5. A variant according to any of the preceding claims selected from the group
consisting of:
(a) .DELTA. 70, 75 - 82;
(b) .DELTA. 75 - 82;
(c) .DELTA. 70, 78, 79;
(d) .DELTA. 70;
(e) .DELTA. 75;
(f) .DELTA. 76;
(g) .DELTA. 78;
(h) .DELTA. 79;
(i) .DELTA. 81; and
(j) .DELTA. 82.




88



6. A variant according to any of the preceding claims further comprising a
substitution of one or more of positions 70 - 84 corresponding to subtilisin
BPN'.
7. A variant according to any of the preceding claims further comprising one
or
more stabilizing mutations.
8. A mutant serine protease gene encoding a variant according to any of the
preceding claims.
9. A personal care composition characterized by:
(a) a variant of a serine protease having a modified amino acid sequence of a
wild-type amino acid sequence, wherein the modified amino acid sequence
comprises a deletion of one or more of positions 70 - 84 corresponding to
BPN'; and
(b) a personal care composition carrier.
10. A composition according to Claim 9 wherein the variant is further
characterized
by a substitution at one or more of positions 70 - 84 corresponding to
subtilisin BPN' .

Description

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SERINE PROTEASE VARIANTS HAVING
AMINO ACID DELETIONS AND SUBSTITUTIONS
FIELD OF THE INVENTION
The present invention relates to serine protease variants and compositions
comprising the variants which have decreased immunogenicity relative to their
corresponding wild-type serine proteases.
BACKGROUND OF THE INVENTION
Enzymes make up the largest class of naturally occurring proteins. One class
of
enzyme includes proteases which catalyze the hydrolysis of other proteins.
This ability
to hydrolyze proteins has been exploited by incorporating naturally occurring
and protein
engineered proteases into cleaning compositions, particularly those relevant
to laundry
applications.
In the cleaning arts, the mostly widely utilized of these proteases are the
serine
proteases. Most of these serine proteases are produced by bacterial organisms
while
some are produced by other organisms, such as fungi. See Siezen, Roland J, et
al.,
"Homology Modelling and Protein Engineering Strategy of Subtilases, the Family
of
Subtilisin-Like Serine Proteases", Protein Engineering, Vol. 4, No. 7, pp. 719
- 737
(1991). Unfortunately, the efficacy of the wild-type proteases in their
natural
environment frequently does not translate into the unnatural cleaning
composition
environment. Specifically, protease characteristics such as, for example,
thermal
stability, pH stability, oxidative stability and substrate specificity are not
necessarily
optimized for utilization outside the natural environment of the enzyme.
Several approaches have been employed to alter the wild-type amino acid
sequence of serine proteases with the goal of increasing the efficacy of the
protease in
the unnatural wash environment. These approaches include the genetic redesign
of
proteases to enhance thermal stability and to improve oxidation stability
under quite
diverse conditions.
However, because such genetically engineered proteases are foreign to mammals,
they are potential antigens. As antigens, these proteases cause immunological
and


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allergic responses (herein collectively described as immunological responses)
in
mammals. In fact, sensitization to serine proteases has been observed in
environments
wherein humans are regularly exposed to the proteases. Such environments
include
manufacturing facilities, where employees are exposed to the proteases through
such
vehicles as uncontrolled dust or aerosolization. Aerosolization can result by
the
introduction of the protease into the lung, which is the route of protease
exposure which
causes the most dangerous response. Protease sensitization can also occur in
the
marketplace, where consumers' repeated use of products containing proteases
may cause
an allergic reaction.
Furthermore, while genetic engineering has been prominent in the continuing
search for more highly effective proteases for use in laundry applications,
genetically
engineered proteases have been minimally utilized in personal care
compositions and
light duty detergents. A primary reason for the absence of engineered
proteases in
products such as, for example, soaps, gels, body washes, and shampoos, is due
to the
aforementioned problem of human sensitization leading to undesirable
immunological
responses. It would therefore be highly advantageous to provide a personal
care
composition which provides the cleansing properties of engineered proteases
with
minimized provocation of immunological responses.
One approach toward alleviating the immunological activity of a protease is
through the redesign of one or more epitopes of the protease. Epitopes are
those amino
acid regions of an antigen which evoke an immunological response through the
binding
of antibodies or the presentation of processed antigens to T cells via a major
histocompatibility complex protein (MHC). Changes in the epitopes can affect
their
efficiency as an antigen. See Walsh, B.J. and M.E.H. Howden, "A Method for the
Detection of IgE Binding Sequences of Allergens Based on a Modification of
Epitope
Mapping", Journal oflmmunological Methods, Vol. 121, pp. 275 - 280 (1989).
The present inventors have discovered that those serine proteases commonly
known as subtilisins, including subtilisin BPN', have a prominent epitope
region at
amino acid positions 70 - 84 corresponding to BPN'. The present inventors have
herein
genetically redesigned such subtilisins to alleviate the immunogenic
properties attributed
to this epitope region. In so doing, the present inventors have discovered
subtilisins


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which evoke a decreased immunological response yet maintain their activity as
an
efficient cleansing protease. Accordingly, the present proteases are suitable
for use in
several types of compositions including, but not limited to, laundry, dish,
hard surface,
skin care, hair care, beauty care, oral, and contact lens compositions.
SUMMARY OF THE INVENTION
The present invention relates to variants of serine proteases having decreased
immunogenicity relative to their corresponding wild-type proteases. More
particularly,
the present invention relates to variants having a modified amino acid
sequence of a
wild-type amino acid sequence, wherein the modified amino acid sequence
comprises a
deletion and, optionally, a substitution of one or more of positions 70 - 84
corresponding
to subtilisin BPN'. The invention further relates to mutant genes encoding
such variants
and cleaning and personal care compositions comprising such variants.
DETAILED DESCRIPTION OF THE INVENTION
The essential components of the present invention are herein described below.
Also included are non-limiting descriptions of various optional and preferred
components useful in embodiments of the present invention.
The present invention can comprise, consist of, or consist essentially of any
of
the required or optional components and / or limitations described herein.
All percentages and ratios are calculated by weight unless otherwise
indicated.
All percentages are calculated based on the total composition unless otherwise
indicated.
All component or composition levels are in reference to the active level of
that
component or composition, and are exclusive of impurities, for example,
residual
solvents or by-products, that may be present in commercially available
sources.
All documents referred to herein, including all patents, patent applications,
and
printed publications, are hereby incorporated by reference in their entirety.
As used herein, abbreviations will be used to describe amino acids. Table I
provides a list of abbreviations used herein:
Table I


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Amino Acid Three-letter AbbreviationOne-letter Abbreviation


Alanine Ala A


Arginine Arg R


Asparagine Asn N


Aspartic Acid Asp D


Cysteine Cys C


Glutamine Gln Q


Glutamic Acid Glu E


Glycine Gly G


Histidine His H


Isoleucine Ile I


Leucine Leu L


Lysine Lys K


Methionine Met M


Phenylalanine Phe F


Proline Pro P


Serine Ser S


Threonine Thr T


Tryptophan Trp W


Tyrosine Tyr Y


Valine Val V


Definitions
As used herein, the term "mutation" refers to alterations in gene sequences
and
amino acid sequences produced by those gene sequences. Mutations may be
deletions,
substitutions, or additions of amino acid residues to the wild-type protein
sequence.
As used herein, the term "wild-type" refers to a protein, herein specifically
a
protease, produced by unmutated organisms.
As used herein, the term "variant" means a protein, herein specifically a
protease,
having an amino acid sequence which differs from that of the wild-type
protease.


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As referred to herein, while the variants of the present invention are not
limited
to those of subtilisin BPN', all amino acid numbering is with reference to the
amino acid
sequence for subtilisin BPN' which is represented by SEQ ID NO:1. The amino
acid
sequence for subtilisin BPN' is further described by Wells, J.A., E. Ferrari,
D.J. Henner,
D.A. Estell, and E.Y. Chen, Nucleic Acids Research, Vol. II, 7911 - 7925
(1983),
incorporated herein by reference.
Variants of the Present Invention
The present inventors have discovered an epitope region in serine proteases
which corresponds to positions 70 - 84 of subtilisin BPN'. The present
inventors have
further discovered that one or more amino acid deletions and / or
substitutions in the
amino acid sequence of a wild-type serine protease provides variants which
evoke a
deceased allergenic and / or immune response relative to the corresponding
wild-type
serine protease.
As used herein, a variant may be designated by referring to the deleted amino
acid positions which characterize the variant. For example, a variant of a
serine protease
having deletions at positions 70, 7S, 76, 77, 78, 79, 80, 81, and 82 may be
designated as
~ 70, 7S - 82. Similarly, substitutions may be indicated by providing the wild-
type
amino acid residue, followed by the position number, followed by the
substituted amino
acid residue to be substituted. Wherein the substituted amino acid residue may
be any
natural amino acid, the symbol "*" is provided. Multiple substitutions
comprising a
variant are separated by the symbol "+". To illustrate, a substitution of
valine for glycine
at position 70 is designated either G1y70Va1 or G70V. A variant comprising
both
deletions and substitutions is designated by combining the aforementioned
designations.
For example, an example of a variant having a substitution at both positions
70 and 72,
as well as deletions at positions 7S - 82 is designated as O 7S - 82, G70V +
V72A.
The variants of the present invention are variants of serine proteases. As
used
herein, the term "serine protease" means a protease which has at least SO%,
and
preferably 80%, amino acid sequence identity with the sequences for one or
more of a
subtilisin-like serine protease. A discussion relating to subtilisin-like
serine proteases
and their homologies may be found in Siezen et al., "Homology Modelling and
Protein


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Engineering Strategy of Subtilases, the Family of Subtilisin-Like Serine
Proteases",
Protein Engineering, Vol. 4, No. 7, pp. 719 - 737 (1991). Preferred serine
proteases for
mutation include subtilisin BPN', subtilisin Carlsberg, subtilisin DY,
subtilisin 309,
proteinase K, and thermitase. The most preferred serine protease for mutation
is
subtilisin BPN'.
The variants of the present invention are variants of serine proteases having
a
modified amino acid sequence of a wild-type amino acid sequence, wherein the
modified
amino acid sequence comprises a deletion of one or more of positions 70, 71,
72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, and 84 (70 - 84) corresponding to BPN'.
Unless
comprising a personal care composition, the variants herein are not a 75 - $3
or a 71.
More preferably, the variants of the present invention comprise a deletion of
one
or more of positions 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, and 83 (73 - 83),
even more
preferably one or more of 75, 76, 77, 78, 79, 80, 81, and 82 (75 - 82).
Preferred variants of the present invention include a 70, 75 - 82; a 75 - 82;
a
70, 78, 79; a 70; a 75; a 76; a 78; a 79; a 81; and a 82. The more preferred
variants
include a 70, 75 - 82; a 75 - 82; a 70, 78, 79; a 70; a 78; and a 79.
The variants of the present invention may optionally, in addition to the one
or
more deletions, further comprise a substitution of one or more of positions 70
- 84
corresponding to subtilisin BPN'. Of course, if a deletion has been made at
any given
position, a substitution cannot be made at that position.
Substitutions in the epitope region are made by replacing the wild-type amino
acid residue with another natural amino acid residue such as one given in
Table I.
Tables 2 - 10 below exemplify non-limiting variants of the present invention.
With respect to these tables, in describing the specific mutations the wild-
type amino
acid residue is given first and the corresponding position number is given
second. Table
2 delineates preferred single deletion variants. Tables 3 - 10 delineate
preferred variants
having one or more deletions and, optionally, one or more substitutions. To
illustrate, the
first example of Table 3 reads "LEU 82 GLY 83". This example is meant to
exemplify
three different variants: one having deletions at positions 82 and 83 (e 82,
83), one .
having a deletion at position 82 and a substitution at 83 (e 82, G83*), and
one having a
substitution at position 82 and a deletion at position 83 (e 83, L82*).


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Table 2
Single Deletion Variants
VAL 84
GLY 83
LEU 82
VAL 81
GLY 80
ILE 79
SER 78
ASN 77
ASN 76
LEU 75
ALA 74
ALA 73
VAL 72
GLY 70
Table 3
Double Mutation Variants
LEU 82 GLY 83
VAL 81 GLY 83
VAL 81 LEU 82
GLY 80 GLY 83
GLY 80 LEU 82
GLY 80 VAL 81
ILE 79 GLY 83
ILE 79 LEU 82
ILE 79 VAL 81
ILE 79 GLY 80
SER 78 GLY 83
SER 78 LEU 82
SER 78 VAL 81
SER 78 GLY 80
SER 78ILE 79


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ASN 77 GLY 83
ASN 77 LEU 82
ASN 77 VAL 81
ASN 7? GLY 80
ASN 77ILE 79
ASN 77 SER 78
ASN 76 GLY 83
ASN 76 LEU 82
ASN 76 VAL 81
ASN 76 GLY 80
ASN 76ILE 79
ASN 76 SER 78
ASN 76 ASN 77
LEU 75 GLY 83
LEU 75 LEU 82
LEU 75 VAL 81
LEU 75 GLY 80
LEU 75ILE 79
LEU 75 SER 78
LEU 75 ASN 77
LEU 75 ASN 76
ALA 74 GLY 83
ALA 74 LEU 82
ALA 74 VAL 81
ALA 74 GLY 80
ALA 74ILE 79
ALA 74 SER 78
ALA 74 ASN 77
ALA 74 ASN 76
ALA 74 LEU 75
ALA 73 GLY 83
ALA 73 LEU 82
ALA 73 VAL 81
ALA 73 GLY 80
ALA 73 ILE 79
ALA 73 SER 78


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ALA 73 ASN 77
ALA 73 ASN 76
ALA 73 LEU 75
ALA 73 ALA 74
Table 4
Triple Mutation Variants
VAL 81 LEU 82 GLY 83
GLY 80 LEU 82 GLY 83
GLY 80 VAL 81 GLY 83
GLY 80 VAL 81 LEU 82
ILE 79 LEU 82 GLY 83
ILE 79 VAL 81 GLY 83
ILE 79 VAL 81 LEU 82
ILE 79 GLY 80 GLY 83
ILE 79 GLY 80 LEU 82
ILE 79 GLY 80 VAL 81
SER 78 LEU 82 GLY 83
SER 78 VAL 81 GLY 83
SER 78 VAL 81 LEU 82
SER 78 GLY 80 GLY 83
SER 78 GLY 80 LEU 82
SER 7$ GLY 80 VAL 81
SER 78 ILE 79 GLY 83
SER 78 ILE 79 LEU 82
SER 78 ILE 79 VAL 81
SER 78 II,E 79 GLY 80
ASN 77 LEU 82 GLY 83
ASN 77 VAL 81 GLY 83
ASN 77 VAL 81 LEU 82
ASN 77 GLY 80 GLY 83
ASN 77 GLY 80 LEU 82
ASN 77 GLY 80 VAL 81
ASN 77 ILE 79 GLY 83
ASN 77 ILE 79 LEU 82
ASN 77 ILE 79 VAL 81


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ASN 77 ILE 79 GLY 80
ASN 77 SER 78 GLY 83
ASN 77 SER 78 LEU 82
ASN 77 SER 78 VAL 81
ASN 77 SER 78 GLY 80
ASN 77 SER 78 ILE 79
ASN 76 LEU 82 GLY 83
ASN 76 VAL 81 GLY 83
ASN 76 VAL 81 LEU 82
ASN 76 GLY 80 GLY 83
ASN 76 GLY 80 LEU 82
ASN 76 GLY 80 VAL 81
ASN 76 ILE 79 GLY 83
ASN 76 ILE 79 LEU 82
ASN 76 ILE 79 VAL 81
ASN 76 ILE 79 GLY 80
ASN 76 SER 78 GLY 83
ASN 76 SER 78 LEU 82
ASN 76 SER 78 VAL 81
ASN 76 SER 78 GLY 80
ASN 76 SER 78 ILE 79
ASN 76 ASN 77 GLY 83
ASN 76 ASN 77 LEU 82
ASN 76 ASN ?7 VAL 81
ASN 76 ASN 77 GLY 80
ASN 76 ASN 77 ILE 79
ASN 76 ASN 77 SER 78
LEU 75 LEU 82 GLY 83
LEU 75 VAL 81 GLY 83
LEU 75 VAL 81 LEU 82
LEU 75 GLY 80 GLY 83
LEU 75 GLY 80 LEU 82
LEU 75 GLY 80 VAL 81
LEU 75 ILE 79 GLY 83
LEU 75 ILE 79 LEU 82
LEU 75 ILE 79 VAL 81


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LEU 75 ILE 79 GLY 80
LEU 75 SER 78 GLY 83
LEU 75 SER 78 LEU 82
LEU 75 SER 78 VAL 81
LEU 75 SER 78 GLY 80
LEU 75 SER 78 ILE 79
LEU 75 ASN 77 GLY 83
LEU 75 ASN 77 LEU 82
LEU 75 ASN 77 VAL 81
LEU ?5 ASN 77 GLY 80
LEU 75 ASN 77 ILE 79
LEU ?5 ASN 77 SER 78
LEU 75 ASN 76 GLY 83
LEU 75 ASN 76 LEU 82
LEU 75 ASN 76 VAL 81
LEU 75 ASN 76 GLY 80
LEU 75 ASN 76 ILE 79
LEU 75 ASN 76 SER 78
LEU 75 ASN 76 ASN 77
ALA 74 LEU 82 GLY 83
ALA 74 VAL 81 GLY 83
ALA 74 VAL 81 LEU 82
ALA 74 GLY 80 GLY 83
ALA 74 GLY 80 LEU 82
ALA 74 GLY 80 VAL 81
ALA 74 ILE 79 GLY 83
ALA 74 ILE 79 LEU 82
ALA 74 ILE 79 VAL 81
ALA 74 ILE 79 GLY 80
ALA 74 SER 78 GLY 83
ALA 74 SER 78 LEU 82
ALA 74 SER 78 VAL 81
ALA 74 SER 78 GLY 80
ALA 74 SER 78 ILE 79
ALA 74 ASN 77 GLY 83
ALA 74 ASN 77 LEU 82


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ALA 74 ASN 7? VAL 81
ALA 74 ASN 77 GLY 80
ALA 74 ASN 77 ILE 79
ALA 74 ASN 77 SER 78
ALA 74 ASN 76 GLY 83
ALA 74 ASN 76 LEU 82
ALA 74 ASN 76 VAL 81
ALA 74 ASN 76 GLY 80
ALA 74 ASN 76 ILE 79
ALA 74 ASN 76 SER 78
ALA 74 ASN 76 ASN 77
ALA 74 LEU 75 GLY 83
ALA 74 LEU 75 LEU 82
ALA 74 LEU 75 VAL 81
ALA 74 LEU 75 GLY 80
ALA 74 LEU 75 ILE 79
ALA 74 LEU 75 SER 78
ALA 74 LEU 75 ASN 77
ALA 74 LEU 75 ASN 76
ALA 73 LEU 82 GLY 83
ALA 73 VAL 81 GLY 83
ALA 73 VAL 81 LEU 82
ALA 73 GLY 80 GLY 83
ALA 73 GLY 80 LEU 82
ALA 73 GLY 80 VAL 81
ALA 73 ILE 79 GLY 83
ALA 73 ILE 79 LEU 82
ALA 731LE 79 VAL 81
ALA 73 ILE 79 GLY 80
ALA 73 SER 78 GLY 83
ALA 73 SER 78 LEU 82
ALA 73 SER 78 VAL 81
ALA 73 SER 78 GLY 80
ALA 73 SER 78 ILE 79
ALA 73 ASN 77 GLY 83
ALA 73 ASN 77 LEU 82


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ALA 73 ASN 77 VAL 81
ALA 73 ASN 77 GLY 80
ALA 73 ASN 77 ILE 79
ALA 73 ASN 77 SER 78
ALA 73 ASN 76 GLY 83
ALA 73 ASN 76 LEU 82
ALA 73 ASN 76 VAL 81
ALA 73 ASN 76 GLY 80
ALA 73 ASN 76 ILE 79
ALA 73 ASN 76 SER 78
ALA 73 ASN 76 ASN 77
ALA 73 LEU 75 GLY 83
ALA 73 LEU 75 LEU 82
ALA 73 LEU 75 VAL 81
ALA 73 LEU 75 GLY 80
ALA 73 LEU 75 ILE 79
ALA 73 LEU 75 SER 78
ALA 73 LEU 75 ASN 77
ALA 73 LEU 75 ASN 76
ALA 73 ALA 74 GLY 83
ALA 73 ALA 74 LEU 82
ALA 73 ALA 74 VAL 81
ALA 73 ALA 74 GLY 80
ALA 73 ALA 74 ILE 79
ALA 73 ALA 74 SER 78
ALA 73 ALA 74 ASN 77
ALA 73 ALA 74 ASN 76
ALA 73 ALA 74 LEU 75
Table 5
Quadruple Mutation Variants
GLY 80 VAL 81 LEU 82 GLY 83
ILE 79 VAL 81 LEU 82 GLY 83
ILE 79 GLY 80 LEU 82 GLY 83
ILE 79 GLY 80 VAL 81 GLY 83


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ILE 79 GLY 80 VAL 81 LEU 82
SER 78 VAL 81 LEU 82 GLY 83
SER 78 GLY 80 LEU 82 GLY 83
SER 78 GLY 80 VAL 81 GLY 83
SER 78 GLY 80 VAL 81 LEU 82
SER 78 ILE 79 LEU 82 GLY 83
SER 78 ILE 79 VAL 81 GLY 83
SER 78 ILE 79 VAL 81 LEU 82
SER 781LE 79 GLY 80 GLY 83
SER 78 ILE 79 GLY 80 LEU 82
SER 78 ILE 79 GLY 80 VAL 81
ASN 77 VAL 81 LEU 82 GLY 83
ASN 77 GLY 80 LEU 82 GLY 83
ASN 77 GLY 80 VAL 81 GLY 83
ASN 77 GLY 80 VAL 81 LEU 82
ASN 77 ILE 79 LEU 82 GLY 83
ASN 77 ILE 79 VAL 81 GLY 83
ASN 77 ILE 79 VAL 81 LEU 82
ASN 77 ILE 79 GLY 80 GLY 83
ASN 77 ILE 79 GLY 80 LEU 82
ASN 77 ILE 79 GLY 80 VAL 81
ASN 77 SER 78 LEU 82 GLY 83
ASN 77 SER 78 VAL 81 GLY 83
ASN 77 SER 78 VAL 81 LEU 82
ASN 77 SER 78 GLY 80 GLY 83
ASN 77 SER 78 GLY 80 LEU 82
ASN 77 SER 78 GLY 80 VAL 81
ASN 77 SER 78 ILE 79 GLY 83
ASN 77 SER 78 ILE 79 LEU 82
ASN 77 SER 78 ILE 79 VAL 81
ASN 77 SER 78 ILE 79 GLY 80
ASN 76 VAL 81 LEU 82 GLY 83
ASN 76 GLY 80 LEU 82 GLY 83
ASN 76 GLY 80 VAL 81 GLY 83
ASN 76 GLY 80 VAL 81 LEU 82
ASN 76 ILE 79 LEU 82 GLY 83


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ASN 76 ILE 79 VAL 81 GLY 83
ASN 76 ILE 79 VAL 81 LEU 82
ASN 76 ILE 79 GLY 80 GLY 83
ASN 76 ILE 79 GLY 80 LEU 82
ASN 76 ILE 79 GLY 80 VAL 81
ASN 76 SER 78 LEU 82 GLY 83
ASN 76 SER 78 VAL 81 GLY 83
ASN 76 SER 78 VAL 81 LEU 82
ASN 76 SER 78 GLY 80 GLY 83
ASN 76 SER 78 GLY 80 LEU 82
ASN 76 SER 78 GLY 80 VAL 81
ASN 76 SER 78 ILE 79 GLY 83
ASN 76 SER 78 ILE 79 LEU 82
ASN 76 SER 78 ILE 79 VAL 81
ASN 76 SER 78 ILE 79 GLY 80
ASN 76 ASN 77 LEU 82 GLY 83
ASN 76 ASN 77 VAL 81 GLY 83
ASN 76 ASN 77 VAL 81 LEU 82
ASN 76 ASN 77 GLY 80 GLY 83
ASN 76 ASN 77 GLY 80 LEU 82
ASN 76 ASN 77 GLY 80 VAL 81
ASN 76 ASN 77 ILE 79 GLY 83
ASN 76 ASN 77 ILE 79 LEU 82
ASN 76 ASN 77 ILE 79 VAL 81
ASN 76 ASN 77 ILE 79 GLY 80
ASN 76 ASN 77 SER 78 GLY 83
ASN 76 ASN 77 SER 78 LEU 82
ASN 76 ASN 77 SER 78 VAL 81
ASN 76 ASN 77 SER 78 GLY 80
ASN 76 ASN 77 SER 78 ILE 79
LEU 75 VAL 81 LEU 82 GLY 83
LEU 75 GLY 80 LEU 82 GLY 83
LEU 75 GLY 80 VAL 81 GLY 83
LEU 75 GLY 80 VAL 81 LEU 82
LEU 75 ILE 79 LEU 82 GLY 83
LEU 75 ILE 79 VAL 81 GLY 83


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LEU 75 ILE 79 VAL 81 LEU 82
LEU 75 ILE 79 GLY 80 GLY 83
LEU 75 ILE 79 GLY 80 LEU 82
LEU 75 ILE 79 GLY 80 VAL 81
LEU 75 SER 78 LEU 82 GLY 83
LEU 75 SER 78 VAL 81 GLY 83
LEU 75 SER 78 VAL 81 LEU 82
LEU 75 SER 78 GLY 80 GLY 83
LEU 75 SER 78 GLY 80 LEU 82
LEU 75 SER 78 GLY 80 VAL 81
LEU 75 SER 78 ILE 79 GLY 83
LEU 75 SER 78 ILE 79 LEU 82
LEU 75 SER 78 ILE 79 VAL 81
LEU 75 SER 78 ILE 79 GLY 80
LEU 75 ASN 77 LEU 82 GLY 83
LEU 75 ASN 77 VAL 81 GLY 83
LEU 75 ASN 77 VAL 81 LEU 82
LEU 75 ASN 77 GLY 80 GLY 83
LEU 75 ASN 77 GLY 80 LEU 82
LEU 75 ASN 77 GLY 80 VAL 81
LEU 75 ASN 77 ILE 79 GLY 83
LEU 75 ASN 77 ILE 79 LEU 82
LEU 75 ASN 77 ILE 79 VAL 81
LEU 75 ASN 77 ILE 79 GLY 80
LEU 75 ASN 77 SER 78 GLY 83
LEU 75 ASN 77 SER 78 LEU 82
LEU 75 ASN 77 SER 78 VAL 81
LEU 75 ASN 77 SER 78 GLY 80
LEU 75 ASN 77 SER 78 ILE 79
LEU 75 ASN 76 LEU 82 GLY 83
LEU 75 ASN 76 VAL 81 GLY 83
LEU 75 ASN 76 VAL 81 LEU 82
LEU 75 ASN 76 GLY 80 GLY 83
LEU 75 ASN 76 GLY 80 LEU 82
LEU 75 ASN 76 GLY 80 VAL 81
LEU 75 ASN 76 ILE 79 GLY 83


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LEU 75 ASN 76 ILE 79 LEU 82
LEU 75 ASN 76 ILE 79 VAL 81
LEU 75 ASN 76 ILE 79 GLY 80
LEU 75 ASN 76 SER 78 GLY 83
LEU 75 ASN 76 SER 78 LEU 82
LEU 75 ASN 76 SER 78 VAL 81
LEU 75 ASN 76 SER 78 GLY 80
LEU 75 ASN 76 SER 78 ILE 79
LEU 75 ASN 76 ASN 77 GLY 83
LEU 75 ASN 76 ASN 77 LEU 82
LEU 75 ASN 76 ASN 77 VAL 81
LEU 75 ASN 76 ASN 77 GLY 80
LEU 75 ASN 76 ASN 77 ILE 79
LEU 75 ASN 76 ASN 77 SER 78
ALA 74 VAL 81 LEU 82 GLY 83
ALA 74 GLY 80 LEU 82 GLY 83
ALA 74 GLY 80 VAL 81 GLY 83
ALA 74 GLY 80 VAL 81 LEU 82
ALA 74 ILE 79 LEU 82 GLY 83
ALA 74 ILE 79 VAL 81 GLY 83
ALA 74 ILE 79 VAL 81 LEU 82
ALA 74 ILE 79 GLY 80 GLY 83
ALA 74 ILE 79 GLY 80 LEU 82
ALA 74 ILE 79 GLY 80 VAL 81
ALA 74 SER 78 LEU 82 GLY 83
ALA 74 SER 78 VAL 8I GLY 83
ALA 74 SER 78 VAL 81 LEU 82
ALA 74 SER 78 GLY 80 GLY 83
ALA 74 SER 78 GLY 80 LEU 82
ALA 74 SER 78 GLY 80 VAL 81
ALA 74 SER 78 ILE 79 GLY 83
ALA 74 SER 78 ILE 79 LEU 82
ALA 74 SER 78 ILE 79 VAL 81
ALA 74 SER 78 ILE 79 GLY 80
ALA 74 ASN 77 LEU 82 GLY 83
ALA 74 ASN 77 VAL 81 GLY 83


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ALA 74 ASN 77 VAL 81 LEU 82
ALA 74 ASN 77 GLY 80 GLY 83
ALA 74 ASN 77 GLY 80 LEU 82
ALA 74 ASN 77 GLY 80 VAL 81
ALA 74 ASN 77 ILE 79 GLY 83
ALA 74 ASN 77 ILE 79 LEU 82
ALA 74 ASN 77 ILE 79 VAL 81
ALA 74 ASN 77 ILE 79 GLY 80
ALA 74 ASN 77 SER 78 GLY 83
ALA 74 ASN 77 SER 78 LEU 82
ALA 74 ASN 77 SER 78 VAL 81
ALA 74 ASN 77 SER 78 GLY 80
ALA 74 ASN 77 SER 78 ILE 79
ALA 74 ASN 76 LEU 82 GLY 83
ALA 74 ASN 76 VAL 81 GLY 83
ALA 74 ASN 76 VAL 81 LEU 82
ALA 74 ASN 76 GLY 80 GLY 83
ALA 74 ASN 76 GLY 80 LEU 82
ALA 74 ASN 76 GLY 80 VAL 8I
ALA 74 ASN 76 ILE 79 GLY 83
ALA 74 ASN 76 ILE 79 LEU 82
ALA 74 ASN 76 ILE 79 VAL 81
ALA 74 ASN 76 ILE 79 GLY 80
ALA 74 ASN 76 SER 78 GLY 83
ALA 74 ASN 76 SER 78 LEU 82
ALA 74 ASN 76 SER 78 VAL 81
ALA 74 ASN 76 SER 78 GLY 80
ALA 74 ASN 76 SER 78 ILE 79
ALA 74 ASN 76 ASN 77 GLY 83
ALA 74 ASN 76 ASN 77 LEU 82
ALA 74 ASN 76 ASN 77 VAL 81
ALA 74 ASN 76 ASN 77 GLY 80
ALA 74 ASN 76 ASN 77 ILE 79
ALA 74 ASN 76 ASN 77 SER 78
ALA 74 LEU 75 LEU 82 GLY 83
ALA 74 LEU 75 VAL 81 GLY 83


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ALA 74 LEU 75 VAL 81 LEU 82
ALA 74 LEU 75 GLY 80 GLY 83
ALA 74 LEU 75 GLY 80 LEU 82
ALA 74 LEU 75 GLY 80 VAL 81
ALA 74 LEU 75 ILE 79 GLY 83
ALA 74 LEU 75 ILE 79 LEU 82
ALA 74 LEU 75 ILE 79 VAL 81
ALA 74 LEU 75 ILE 79 GLY 80
ALA 74 LEU 75 SER 78 GLY 83
ALA 74 LEU 75 SER 78 LEU 82
ALA 74 LEU 75 SER 78 VAL 81
ALA 74 LEU 75 SER 78 GLY 80
ALA 74 LEU 75 SER 78 ILE 79
ALA 74 LEU 75 ASN 77 GLY 83
ALA 74 LEU 75 ASN 77 LEU 82
ALA 74 LEU 75 ASN 77 VAL 81
ALA 74 LEU ?5 ASN 77 GLY 80
ALA 74 LEU 75 ASN 77 ILE 79
ALA 74 LEU 75 ASN 77 SER 78
ALA 74 LEU 75 ASN 76 GLY 83
ALA 74 LEU 75 ASN 76 LEU 82
ALA 74 LEU 75 ASN 76 VAL 81
ALA 74 LEU 75 ASN 76 GLY 80
ALA 74 LEU 75 ASN 76 ILE 79
ALA 74 LEU 75 ASN 76 SER 78
ALA 74 LEU 75 ASN 76 ASN 77
ALA 73 VAL 81 LEU 82 GLY 83
ALA 73 GLY 80 LEU 82 GLY 83
ALA 73 GLY 80 VAL 81 GLY 83
ALA 73 GLY 80 VAL 8 t LEU 82
ALA 73 ILE 79 LEU 82 GLY 83
ALA 73 ILE 79 VAL 81 GLY 83
ALA 73 ILE 79 VAL 81 LEU 82
ALA 73 ILE 79 GLY 80 GLY 83
ALA 73 ILE 79 GLY 80 LEU 82
ALA 73 ILE 79 GLY 80 VAL 81


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ALA 73 SER 78 LEU 82 GLY 83
ALA 73 SER 78 VAL 81 GLY 83
ALA 73 SER 78 VAL 81 LEU 82
ALA 73 SER 78 GLY 80 GLY 83
ALA 73 SER 78 GLY 80 LEU 82
ALA 73 SER 78 GLY 80 VAL 81
ALA 73 SER 78 ILE 79 GLY 83
ALA 73 SER 78 ILE 79 LEU 82
ALA 73 SER 78 ILE 79 VAL 81
ALA 73 SER 78 ILE 79 GLY 80
ALA 73 ASN 77 LEU 82 GLY 83
ALA 73 ASN 77 VAL 81 GLY 83
ALA 73 ASN 77 VAL 81 LEU 82
ALA 73 ASN 77 GLY 80 GLY 83
ALA 73 ASN 77 GLY 80 LEU 82
ALA 73 ASN 77 GLY 80 VAL 81
ALA 73 ASN 77 ILE 79 GLY 83
ALA 73 ASN 77 ILE 79 LEU 82
ALA 73 ASN 77 ILE 79 VAL 81
ALA 73 ASN 77 ILE 79 GLY 80
ALA 73 ASN 77 SER 78 GLY 83
ALA 73 ASN 77 SER 78 LEU 82
ALA 73 ASN 77 SER 78 VAL 81
ALA 73 ASN 77 SER 78 GLY 80
ALA 73 ASN 77 SER 78 ILE 79
ALA 73 ASN 76 LEU 82 GLY 83
ALA 73 ASN 76 VAL 81 GLY 83
ALA 73 ASN 76 VAL 81 LEU 82
ALA 73 ASN 76 GLY 80 GLY 83
ALA 73 ASN 76 GLY 80 LEU 82
ALA 73 ASN 76 GLY 80 VAL 81
ALA 73 ASN 76 ILE 79 GLY 83
ALA 73 ASN 76 ILE 79 LEU 82
ALA 73 ASN 76 ILE 79 VAL 81
ALA 73 ASN 76 ILE 79 GLY 80
ALA 73 ASN 76 SER 78 GLY 83


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ALA 73 ASN 76 SER 78 LEU 82
ALA 73 ASN 76 SER 78 VAL 81
ALA 73 ASN 76 SER 78 GLY 80
ALA 73 ASN 76 SER 78 ILE 79
ALA 73 ASN 76 ASN 77 GLY 83
ALA 73 ASN 76 ASN 77 LEU 82
ALA 73 ASN 76 ASN 77 VAL 81
ALA 73 ASN 76 ASN 77 GLY 80
ALA 73 ASN 76 ASN 77 ILE 79
ALA 73 ASN 76 ASN 77 SER 78
ALA 73 LEU 75 LEU 82 GLY 83
ALA 73 LEU 75 VAL 81 GLY 83
ALA 73 LEU 75 VAL 81 LEU 82
ALA 73 LEU 75 GLY 80 GLY 83
ALA 73 LEU 75 GLY 80 LEU 82
ALA 73 LEU 75 GLY 80 VAL 81
ALA 73 LEU ?5 ILE 79 GLY 83
ALA 73 LEU 75 ILE 79 LEU 82
ALA 73 LEU 75 ILE 79 VAL 81
ALA 73 LEU 75 ILE 79 GLY 80
ALA 73 LEU 75 SER 78 GLY 83
ALA 73 LEU 75 SER 78 LEU 82
ALA 73 LEU 75 SER 78 VAL 81
ALA 73 LEU 75 SER 78 GLY 80
ALA 73 LEU 75 SER 78 ILE 79
ALA 73 LEU 75 ASN 77 GLY 83
ALA 73 LEU 75 ASN 77 LEU 82
ALA 73 LEU 75 ASN 77 VAL 81
ALA 73 LEU 75 ASN 77 GLY 80
ALA 73 LEU 75 ASN 77 ILE 79
ALA 73 LEU 75 ASN 77 SER 78
ALA 73 LEU 75 ASN 76 GLY 83
ALA 73 LEU 75 ASN 76 LEU 82
ALA 73 LEU 75 ASN 76 VAL 81
ALA 73 LEU 75 ASN 76 GLY 80
ALA 73 LEU 75 ASN 76 ILE 79


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ALA 73 LEU 75 ASN 76 SER 78
ALA 73 LEU 75 ASN 76 ASN 77
ALA 73 ALA 74 LEU 82 GLY 83
ALA 73 ALA 74 VAL 81 GLY 83
ALA 73 ALA 74 VAL 81 LEU 82
ALA 73 ALA 74 GLY 80 GLY 83
ALA 73 ALA 74 GLY 80 LEU 82
ALA 73 ALA 74 GLY 80 VAL 81
ALA 73 ALA 74 ILE 79 GLY 83
ALA 73 ALA 74 ILE 79 LEU 82
ALA 73 ALA 74 ILE 79 VAL 81
ALA 73 ALA 74 ILE 79 GLY 80
ALA 73 ALA 74 SER 78 GLY 83
ALA 73 ALA 74 SER 78 LEU 82
ALA 73 ALA 74 SER 78 VAL 81
ALA 73 ALA 74 SER' 78 GLY 80
ALA 73 ALA 74 SER 78 ILE 79
ALA 73 ALA 74 ASN 77 GLY 83
ALA 73 ALA 74 ASN 77 LEU 82
ALA 73 ALA 74 ASN 77 VAL 81
ALA 73 ALA 74 ASN 77 GLY 80
ALA 73 ALA 74 ASN 77 ILE 79
ALA 73 ALA 74 ASN 77 SER 78
ALA 73 ALA 74 ASN 76 GLY 83
ALA 73 ALA 74 ASN 76 LEU 82
ALA 73 ALA 74 ASN ?6 VAL 81
ALA 73 ALA 74 ASN 76 GLY 80
ALA 73 ALA 74 ASN 76 ILE 79
ALA 73 ALA 74 ASN 76 SER 78
ALA 73 ALA 74 ASN 76 ASN 77
ALA 73 ALA 74 LEU 75 GLY 83
ALA 73 ALA 74 LEU 75 LEU 82
ALA 73 ALA 74 LEU 75 VAL 81
ALA 73 ALA 74 LEU 75 GLY 80
ALA 73 ALA 74 LEU 75 ILE 79
ALA 73 ALA 74 LEU 75 SER 78


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ALA 73 ALA 74 LEU 75 ASN 77
ALA 73 ALA 74 LEU 75 ASN 76
Table 6
Quintuple Mutation Variants
ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
SER 78 GLY 80 VAL 81 LEU 82 GLY 83
SER 78 ILE 79 VAL 81 LEU 82 GLY 83
SER 78 ILE 79 GLY 80 LEU 82 GLY 83
SER 78 ILE 79 GLY 80 VAL 81 GLY 83
SER 78 ILE 79 GLY 80 VAL 81 LEU 82
ASN 77 GLY 80 VAL 81 LEU 82 GLY 83
ASN 77 ILE 79 VAL 81 LEU 82 GLY 83
ASN 77 ILE 79 GLY 80 LEU 82 GLY 83
ASN 77 ILE 79 GLY 80 VAL 81 GLY 83
ASN 77 ILE 79 GLY 80 VAL 81 LEU 82
ASN 77 SER 78 VAL 81 LEU 82 GLY 83
ASN 77 SER 78 GLY 80 LEU 82 GLY 83
ASN 77 SER 78 GLY 80 VAL 81 GLY 83
ASN 77 SER 78 GLY 80 VAL 81 LEU 82
ASN 77 SER 78 ILE 79 LEU 82 GLY 83
ASN 77 SER 78 ILE 79 VAL 81 GLY 83
ASN 77 SER 78 ILE 79 VAL 81 LEU 82
ASN 77 SER 78 ILE 79 GLY 80 GLY 83
ASN 77 SER 78 ILE 79 GLY 80 LEU 82
ASN 77 SER 78 ILE 79 GLY 80 VAL 81
ASN 76 GLY 80 VAL 81 LEU 82 GLY 83
ASN 76 ILE 79 VAL 81 LEU 82 GLY 83
ASN 76 ILE 79 GLY 80 LEU 82 GLY 83
ASN 76 ILE 79 GLY 80 VAL 81 GLY 83
ASN 76 ILE 79 GLY 80 VAL 81 LEU 82
ASN ?6 SER 78 VAL 81 LEU 82 GLY 83
ASN 76 SER 78 GLY 80 LEU 82 GLY 83
ASN 76 SER 78 GLY 80 VAL 81 GLY 83
ASN 76 SER 78 GLY 80 VAL 81 LEU 82
ASN 76 SER 78 ILE 79 LEU 82 GLY 83


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ASN 76 SER 78 ILE 79 VAL 81 GLY 83
ASN 76 SER 78 ILE 79 VAL 81 LEU 82
ASN 76 SER 78 ILE 79 GLY 80 GLY 83
ASN 76 SER 78 ILE 79 GLY 80 LEU 82
ASN 76 SER 78 ILE 79 GLY 80 VAL 81
ASN 76 ASN 77 VAL 81 LEU 82 GLY 83
ASN 76 ASN 77 GLY 80 LEU 82 GLY 83
ASN 76 ASN 77 GLY 80 VAL 81 GLY 83
ASN 76 ASN 77 GLY 80 VAL 81 LEU 82
ASN 76 ASN 77 ILE 79 LEU 82 GLY 83
ASN 76 ASN 77 ILE 79 VAL 81 GLY 83
ASN 76 ASN 771LE 79 VAL 81 LEU 82
ASN 76 ASN 77 ILE 79 GLY 80 GLY 83
ASN 76 ASN 77 ILE 79 GLY 80 LEU 82
ASN 76 ASN 77 ILE 79 GLY 80 VAL 81
ASN 76 ASN 77 SER 78 LEU 82 GLY 83
ASN 76 ASN 77 SER 78 VAL 81 GLY 83
ASN 76 ASN 77 SER 78 VAL 81 LEU 82
ASN 76 ASN 77 SER 78 GLY 80 GLY 83
ASN 76 ASN 77 SER 78 GLY 80 LEU 82
ASN 76 ASN 77 SER 78 GLY 80 VAL 81
ASN 76 ASN 77 SER 78 ILE 79 GLY 83
ASN 76 ASN 77 SER 78 ILE 79 LEU 82
ASN 76 ASN 77 SER 78 ILE 79 VAL 81
ASN 76 ASN 77 SER 78 ILE 79 GLY 80
LEU 75 GLY 80 VAL 81 LEU 82 GLY 83
LEU 75 ILE ?9 VAL 81 LEU 82 GLY 83
LEU 75 ILE 79 GLY 80 LEU 82 GLY 83
LEU 75 ILE 79 GLY 80 VAL 8I GLY 83
LEU 75 ILE 79 GLY 80 VAL 81 LEU 82
LEU 75 SER 78 VAL 81 LEU 82 GLY 83
LEU 75 SER 78 GLY 80 LEU 82 GLY 83
LEU 75 SER 78 GLY 80 VAL 81 GLY 83
LEU 75 SER 78 GLY 80 VAL 81 LEU 82
LEU 75 SER 78 ILE 79 LEU 82 GLY 83
LEU 75 SER 78 ILE 79 VAL 81 GLY 83


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LEU 75 SER 78 ILE 79 VAL 81 LEU 82
LEU 75 SER 78 ILE 79 GLY 80 GLY 83
LEU 75 SER 78 ILE 79 GLY 80 LEU 82
LEU 75 SER 78 ILE 79 GLY 80 VAL 81
LEU 75 ASN 77 VAL 81 LEU 82 GLY 83
LEU 75 ASN 77 GLY 80 LEU 82 GLY 83
LEU 75 ASN 77 GLY 80 VAL 81 GLY 83
LEU 75 ASN 77 GLY 80 VAL 81 LEU 82
LEU 75 ASN 77 ILE 79 LEU 82 GLY 83
LEU 75 ASN 77 ILE 79 VAL 81 GLY 83
LEU 75 ASN 77 ILE 79 VAL 81 LEU 82
LEU 75 ASN 77 ILE 79 GLY 80 GLY 83
LEU 75 ASN 77 ILE 79 GLY 80 LEU 82
LEU 75 ASN 77 ILE 79 GLY 80 VAL 81
LEU 75 ASN 77 SER 78 LEU 82 GLY 83
LEU 75 ASN 77 SER 78 VAL 81 GLY 83
LEU 75 ASN 77 SER 78 VAL 81 LEU 82
LEU 75 ASN 77 SER 78 GLY 80 GLY 83
LEU 75 ASN 77 SER 78 GLY 80 LEU 82
LEU 75 ASN 77 SER 78 GLY 80 VAL 81
LEU 75 ASN 77 SER 78 ILE 79 GLY 83
LEU 75 ASN 77 SER 78 ILE 79 LEU 82
LEU 75 ASN 77 SER 78 ILE 79 VAL 81
LEU 75 ASN 77 SER 78 ILE 79 GLY 80
LEU 75 ASN 76 VAL 81 LEU 82 GLY 83
LEU 75 ASN 76 GLY 80 LEU 82 GLY 83
LEU 75 ASN 76 GLY 80 VAL 81 GLY 83
LEU 75 ASN 76 GLY 80 VAL 81 LEU 82
LEU 75 ASN 76 ILE 79 LEU 82 GLY 83
LEU 75 ASN 76 ILE 79 VAL 81 GLY 83
LEU 75 ASN 76 ILE 79 VAL 81 LEU 82
LEU 75 ASN 76 ILE 79 GLY 80 GLY 83
LEU 75 ASN 76 ILE 79 GLY 80 LEU 82
LEU 75 ASN 76 ILE 79 GLY 80 VAL 81
LEU 75 ASN 76 SER 78 LEU 82 GLY 83
LEU 75 ASN 76 SER 78 VAL 81 GLY 83


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LEU 75 ASN 76 SER 78 VAL 81 LEU 82
LEU 75 ASN 76 SER 78 GLY 80 GLY 83
LEU 75 ASN 76 SER 78 GLY 80 LEU 82
LEU 75 ASN 76 SER 78 GLY 80 VAL 81
LEU 75 ASN 76 SER 78 ILE 79 GLY 83
LEU 75 ASN 76 SER 78 ILE 79 LEU 82
LEU 75 ASN 76 SER 78 ILE 79 VAL 81
LEU 75 ASN 76 SER 78 ILE 79 GLY 80
LEU 75 ASN 76 ASN 77 LEU 82 GLY 83
LEU 75 ASN 76 ASN 77 VAL 81 GLY 83
LEU 75 ASN 76 ASN 77 VAL 81 LEU 82
LEU 75 ASN 76 ASN 77 GLY 80 GLY 83
LEU 75 ASN 76 ASN 77 GLY 80 LEU 82
LEU 75 ASN 76 ASN 77 GLY 80 VAL 81
LEU 75 ASN 76 ASN 77 ILE 79 GLY 83
LEU 75 ASN 76 ASN 77 ILE 79 LEU 82
LEU 75 ASN 76 ASN 77 ILE 79 VAL 81
LEU 75 ASN 76 ASN 77 ILE 79 GLY 80
LEU 75 ASN 76 ASN 77 SER 78 GLY 83
LEU 75 ASN 76 ASN 77 SER 78 LEU 82
LEU 75 ASN 76 ASN 77 SER 78 VAL 81
LEU 75 ASN 76 ASN 77 SER 78 GLY 80
LEU 75 ASN 76 ASN 77 SER 78 ILE 79
ALA 74 GLY 80 VAL 81 LEU 82 GLY 83
ALA 74 ILE 79 VAL 81 LEU 82 GLY 83
ALA 74 ILE 79 GLY 80 LEU 82 GLY 83
ALA 74 ILE 79 GLY 80 VAL 81 GLY 83
ALA 74 ILE 79 GLY 80 VAL 81 LEU 82
ALA 74 SER 78 VAL 81 LEU 82 GLY 83
ALA 74 SER 78 GLY 80 LEU 82 GLY 83
ALA 74 SER 78 GLY 80 VAL 81 GLY 83
ALA 74 SER 78 GLY 80 VAL 81 LEU 82
ALA 74 SER 78 ILE 79 LEU 82 GLY 83
ALA 74 SER 78 ILE 79 VAL 81 GLY 83
ALA 74 SER 78 ILE 79 VAL 81 LEU 82
ALA 74 SER 78 ILE 79 GLY 80 GLY 83


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ALA 74 SER 78 ILE 79 GLY 80 LEU 82
ALA 74 SER 78 ILE 79 GLY 80 VAL 81
ALA 74 ASN 77 VAL 81 LEU 82 GLY 83
ALA 74 ASN 77 GLY 80 LEU 82 GLY 83
ALA 74 ASN 77 GLY 80 VAL 81 GLY 83
ALA 74 ASN 77 GLY 80 VAL 81 LEU 82
ALA 74 ASN 77 ILE 79 LEU 82 GLY 83
ALA 74 ASN 77 ILE 79 VAL 81 GLY 83
ALA 74 ASN 77 ILE 79 VAL 81 LEU 82
ALA 74 ASN 77 ILE 79 GLY 80 GLY 83
ALA 74 ASN 77 ILE 79 GLY 80 LEU 82
ALA 74 ASN 77 ILE 79 GLY 80 VAL 81
ALA 74 ASN 77 SER 78 LEU 82 GLY 83
ALA 74 ASN 77 SER 78 VAL 81 GLY 83
ALA 74 ASN 77 SER 78 VAL 81 LEU 82
ALA 74 ASN 77 SER 78 GLY 80 GLY 83
ALA 74 ASN 77 SER 78 GLY 80 LEU 82
ALA 74 ASN 77 SER 78 GLY 80 VAL 81
ALA 74 ASN 77 SER 78 ILE 79 GLY 83
ALA 74 ASN 77 SER 78 ILE 79 LEU 82
ALA 74 ASN 77 SER 78 ILE 79 VAL 81
ALA 74 ASN 77 SER 78 ILE 79 GLY 80
ALA 74 ASN 76 VAL 81 LEU 82 GLY 83
ALA 74 ASN 76 GLY 80 LEU 82 GLY 83
ALA 74 ASN 76 GLY 80 VAL 81 GLY 83
ALA 74 ASN 76 GLY 80 VAL 81 LEU 82
ALA 74 ASN 76 ILE 79 LEU 82 GLY 83
ALA 74 ASN 76 ILE 79 VAL 81 GLY 83
ALA 74 ASN 76 ILE 79 VAL 81 LEU 82
ALA 74 ASN 76 ILE 79 GLY 80 GLY 83
ALA 74 ASN 76 ILE 79 GLY 80 LEU 82
ALA 74 ASN 76 ILE 79 GLY 80 VAL 81
ALA 74 ASN 76 SER 78 LEU 82 GLY 83
ALA 74 ASN 76 SER 78 VAL 81 GLY 83
ALA 74 ASN 76 SER 78 VAL 81 LEU 82
ALA 74 ASN 76 SER 78 GLY 80 GLY 83


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ALA 74 ASN 76 SER 78 GLY 80 LEU 82
ALA 74 ASN 76 SER 78 GLY 80 VAL 81
ALA 74 ASN 76 SER 78 ILE 79 GLY 83
ALA 74 ASN 76 SER 78 ILE 79 LEU 82
ALA 74 ASN 76 SER 78 ILE 79 VAL 81
ALA 74 ASN 76 SER 78 ILE 79 GLY 80
ALA 74 ASN 76 ASN 77 LEU 82 GLY 83
ALA 74 ASN 76 ASN 77 VAL 81 GLY 83
ALA 74 ASN 76 ASN 77 VAL 81 LEU 82
ALA 74 ASN 76 ASN 77 GLY 80 GLY 83
ALA 74 ASN 76 ASN 77 GLY 80 LEU 82
ALA 74 ASN 76 ASN 77 GLY 80 VAL 81
ALA 74 ASN 76 ASN 77 ILE 79 GLY 83
ALA 74 ASN 76 ASN 77 ILE 79 LEU 82
ALA 74 ASN 76 ASN 77 ILE 79 VAL 81
ALA 74 ASN 76 ASN 77 ILE 79 GLY 80
ALA 74 ASN 76 ASN 77 SER 78 GLY 83
ALA 74 ASN 76 ASN 77 SER 78 LEU 82
ALA 74 ASN 76 ASN 77 SER 78 VAL 81
ALA 74 ASN 76 ASN 77 SER 78 GLY 80
ALA 74 ASN 76 ASN 77 SER 78 ILE 79
ALA 74 LEU 75 VAL 81 LEU 82 GLY 83
ALA 74 LEU 75 GLY 80 LEU 82 GLY 83
ALA 74 LEU 75 GLY 80 VAL 81 GLY 83
ALA 74 LEU 75 GLY 80 VAL 81 LEU 82
ALA 74 LEU 75 ILE 79 LEU 82 GLY 83
ALA 74 LEU 75 ILE 79 VAL 81 GLY 83
ALA 74 LEU 75 ILE 79 VAL 81 LEU 82
ALA 74 LEU 75 ILE 79 GLY 80 GLY 83
ALA 74 LEU 75 ILE 79 GLY 80 LEU 82
ALA 74 LEU 75 ILE 79 GLY 80 VAL 81
ALA 74 LEU 75 SER 78 LEU 82 GLY 83
ALA 74 LEU 75 SER 78 VAL 81 GLY 83
ALA 74 LEU 75 SER 78 VAL 81 LEU 82
ALA 74 LEU 75 SER 78 GLY 80 GLY 83
ALA 74 LEU 75 SER 78 GLY 80 LEU 82


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ALA 74 LEU 75 SER 78 GLY 80 VAL 81
ALA 74 LEU 75 SER 78 ILE 79 GLY 83
ALA 74 LEU 75 SER 78 ILE 79 LEU 82
ALA 74 LEU 75 SER 78 ILE 79 VAL 81
ALA 74 LEU 75 SER 78 ILE 79 GLY 80
ALA 74 LEU 75 ASN 77 LEU 82 GLY 83
ALA 74 LEU 75 ASN 77 VAL 81 GLY 83
ALA 74 LEU 75 ASN 77 VAL 81 LEU 82
ALA 74 LEU 75 ASN 77 GLY 80 GLY 83
ALA 74 LEU 75 ASN 77 GLY 80 LEU 82
ALA 74 LEU 75 ASN 77 GLY 80 VAL 81
ALA 74 LEU 75 ASN 77 ILE 79 GLY 83
ALA 74 LEU 75 ASN 77 ILE 79 LEU 82
ALA 74 LEU 75 ASN 77 ILE 79 VAL 81
ALA 74 LEU 75 ASN 77 ILE 79 GLY 80
ALA 74 LEU 75 ASN 77 SER 78 GLY 83
ALA 74 LEU 75 ASN 77 SER 78 LEU 82
ALA 74 LEU 75 ASN 77 SER 78 VAL 81
ALA 74 LEU 75 ASN 77 SER 78 GLY 80
ALA 74 LEU 75 ASN 77 SER 78 ILE 79
ALA 74 LEU 75 ASN 76 LEU 82 GLY 83
ALA 74 LEU 75 ASN 76 VAL 81 GLY 83
ALA 74 LEU 75 ASN 76 VAL 81 LEU 82
ALA 74 LEU 75 ASN 76 GLY 80 GLY 83
ALA 74 LEU 75 ASN 76 GLY 80 LEU 82
ALA 74 LEU 75 ASN 76 GLY 80 VAL 81
ALA 74 LEU 75 ASN 76 ILE 79 GLY 83
ALA 74 LEU 75 ASN 76 ILE 79 LEU 82
ALA 74 LEU 75 ASN 76 ILE 79 VAL 81
ALA 74 LEU 75 ASN 76 ILE 79 GLY 80
ALA 74 LEU 75 ASN 76 SER 78 GLY 83
ALA 74 LEU 75 ASN 76 SER 78 LEU 82
ALA 74 LEU 75 ASN 76 SER 78 VAL 81
ALA 74 LEU 75 ASN 76 SER 78 GLY 80
ALA 74 LEU 75 ASN 76 SER 78 ILE 79
ALA 74 LEU 75 ASN 76 ASN 77 GLY 83


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ALA 74 LEU 75 ASN 76 ASN 77 LEU 82
ALA 74 LEU 75 ASN 76 ASN 77 VAL 81
ALA 74 LEU 75 ASN 76 ASN 77 GLY 80
ALA 74 LEU 75 ASN 76 ASN 77 ILE 79
ALA 74 LEU 75 ASN 76 ASN 77 SER 78
ALA 73 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ILE 79 VAL 81 LEU 82 GLY 83
ALA 73 ILE 79 GLY 80 LEU 82 GLY 83
ALA 73 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 ILE 79 GLY 80 VAL 81 LEU 82
ALA 73 SER 78 VAL 81 LEU 82 GLY 83
ALA 73 SER 78 GLY 80 LEU 82 GLY 83
ALA 73 SER 78 GLY 80 VAL 81 GLY 83
ALA 73 SER 78 GLY 80 VAL 81 LEU 82
ALA 73 SER 78 ILE 79 LEU 82 GLY 83
ALA 73 SER 78 ILE 79 VAL 81 GLY 83
ALA 73 SER 78 ILE 79 VAL 81 LEU 82
ALA 73 SER 78 ILE 79 GLY 80 GLY 83
ALA 73 SER 78 ILE 79 GLY 80 LEU 82
ALA 73 SER 78 ILE 79 GLY 80 VAL 81
ALA 73 ASN 77 VAL 81 LEU 82 GLY 83
ALA 73 ASN 77 GLY 80 LEU 82 GLY 83
ALA 73 ASN 77 GLY 80 VAL 81 GLY 83
ALA 73 ASN 77 GLY 80 VAL 81 LEU 82
ALA 73 ASN 77 ILE 79 LEU 82 GLY 83
ALA 73 ASN 77 ILE 79 VAL 81 GLY 83
ALA 73 ASN 77 ILE 79 VAL 81 LEU 82
ALA 73 ASN 77 ILE 79 GLY 80 GLY 83
ALA 73 ASN 77 ILE 79 GLY 80 LEU 82
ALA 73 ASN 77 ILE 79 GLY 80 VAL 81
ALA 73 ASN 77 SER 78 LEU 82 GLY 83
ALA 73 ASN 77 SER 78 VAL 81 GLY 83
ALA 73 ASN 77 SER 78 VAL 81 LEU 82
ALA 73 ASN 77 SER 78 GLY 80 GLY 83
ALA 73 ASN 77 SER 78 GLY 80 LEU 82
ALA 73 ASN 77 SER 78 GLY 80 VAL 81


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ALA 73 ASN 77 SER 78 ILE 79 GLY 83
ALA 73 ASN 77 SER 78 ILE 79 LEU 82
ALA 73 ASN 77 SER 78 ILE 79 VAL 81
ALA 73 ASN 77 SER 78 ILE 79 GLY 80
ALA 73 ASN 76 VAL 81 LEU 82 GLY 83
ALA 73 ASN 76 GLY 80 LEU 82 GLY 83
ALA 73 ASN 76 GLY 80 VAL 81 GLY 83
ALA 73 ASN 76 GLY 80 VAL 81 LEU 82
ALA 73 ASN 76 ILE 79 LEU 82 GLY 83
ALA 73 ASN 76 ILE 79 VAL 81 GLY 83
ALA 73 ASN 76 ILE 79 VAL 81 LEU 82
ALA 73 ASN 76 ILE 79 GLY 80 GLY 83
ALA 73 ASN 76 ILE 79 GLY 80 LEU 82
ALA 73 ASN 76 ILE 79 GLY 80 VAL 81
ALA 73 ASN 76 SER 78 LEU 82 GLY 83
ALA 73 ASN 76 SER 78 VAL 81 GLY 83
ALA 73 ASN 76 SER 78 VAL 81 LEU 82
ALA 73 ASN 76 SER 78 GLY 80 GLY 83
ALA 73 ASN 76 SER 78 GLY 80 LEU 82
ALA 73 ASN 76 SER 78 GLY 80 VAL 81
ALA 73 ASN 76 SER 78 ILE 79 GLY 83
ALA 73 ASN 76 SER 78 ILE 79 LEU 82
ALA 73 ASN 76 SER 78 ILE 79 VAL 81
ALA 73 ASN 76 SER 78 ILE 79 GLY 80
ALA 73 ASN 76 ASN 77 LEU 82 GLY 83
ALA 73 ASN 76 ASN 77 VAL 81 GLY 83
ALA 73 ASN 76 ASN 77 VAL 81 LEU 82
ALA 73 ASN 76 ASN 77 GLY 80 GLY 83
ALA 73 ASN 76 ASN 77 GLY 80 LEU 82
ALA 73 ASN 76 ASN 77 GLY 80 VAL 81
ALA 73 ASN 76 ASN 77 ILE 79 GLY 83
ALA 73 ASN 76 ASN 77 ILE 79 LEU 82
ALA 73 ASN 76 ASN 77 ILE 79 VAL 81
ALA 73 ASN 76 ASN 77 ILE 79 GLY 80
ALA 73 ASN 76 ASN 77 SER 78 GLY 83
ALA 73 ASN 76 ASN 77 SER 78 LEU 82


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ALA 73 ASN 76 ASN 77 SER 78 VAL 81
ALA 73 ASN 76 ASN 77 SER 78 GLY 80
ALA 73 ASN 76 ASN 77 SER 78 ILE 79
ALA 73 LEU 75 VAL 81 LEU 82 GLY 83
ALA 73 LEU 75 GLY 80 LEU 82 GLY 83
ALA 73 LEU 75 GLY 80 VAL 81 GLY 83
ALA 73 LEU 75 GLY 80 VAL 81 LEU 82
ALA 73 LEU 75 ILE 79 LEU 82 GLY 83
ALA 73 LEU 75 ILE 79 VAL 81 GLY 83
ALA 73 LEU 75 ILE 79 VAL 81 LEU 82
ALA 73 LEU 75 ILE 79 GLY 80 GLY 83
ALA 73 LEU 75 ILE 79 GLY 80 LEU 82
ALA 73 LEU 75 ILE 79 GLY 80 VAL 81
ALA 73 LEU 75 SER 78 LEU 82 GLY 83
ALA 73 LEU 75 SER 78 VAL 81 GLY 83
ALA 73 LEU 75 SER 78 VAL 81 LEU 82
ALA 73 LEU 75 SER 78 GLY 80 GLY 83
ALA 73 LEU 75 SER 78 GLY 80 LEU 82
ALA 73 LEU ?5 SER 78 GLY 80 VAL 81
ALA 73 LEU 75 SER 78 ILE 79 GLY 83
ALA 73 LEU 75 SER 78 ILE 79 LEU 82
ALA 73 LEU 75 SER 78 ILE 79 VAL 81
ALA 73 LEU 75 SER 78 ILE 79 GLY 80
ALA 73 LEU 75 ASN 77 LEU 82 GLY 83
ALA 73 LEU 75 ASN 77 VAL 81 GLY 83
ALA 73 LEU 75 ASN 77 VAL 81 LEU 82
ALA 73 LEU 75 ASN 77 GLY 80 GLY 83
ALA 73 LEU 75 ASN 77 GLY 80 LEU 82
ALA 73 LEU 75 ASN 77 GLY 80 VAL 81
ALA 73 LEU 75 ASN 77 ILE 79 GLY 83
ALA 73 LEU 75 ASN 77 ILE 79 LEU 82
ALA 73 LEU 75 ASN 77 ILE 79 VAL 81
ALA 73 LEU 75 ASN 77 ILE 79 GLY 80
ALA 73 LEU 75 ASN 77 SER 78 GLY 83
ALA 73 LEU 75 ASN 77 SER 78 LEU 82
ALA 73 LEU ?5 ASN 77 SER 78 VAL 81


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ALA 73 LEU 75 ASN 77 SER 78 GLY 80
ALA 73 LEU 75 ASN 77 SER 78 ILE 79
ALA 73 LEU 75 ASN 76 LEU 82 GLY 83
ALA 73 LEU 75 ASN 76 VAL 81 GLY 83
ALA 73 LEU 75 ASN 76 VAL 81 LEU 82
ALA 73 LEU 75 ASN 76 GLY 80 GLY 83
ALA 73 LEU 75 ASN 76 GLY 80 LEU 82
ALA 73 LEU 75 ASN 76 GLY 80 VAL 81
ALA 73 LEU 75 ASN 76 ILE 79 GLY 83
ALA 73 LEU 75 ASN 76 ILE 79 LEU 82
ALA 73 LEU 75 ASN 76 ILE 79 VAL 81
ALA 73 LEU 75 ASN 76 ILE 79 GLY 80
ALA 73 LEU 75 ASN 76 SER 78 GLY 83
ALA 73 LEU 75 ASN 76 SER 78 LEU 82
ALA 73 LEU 75 ASN 76 SER 78 VAL 81
ALA 73 LEU 75 ASN 76 SER 78 GLY 80
ALA 73 LEU 75 ASN 76 SER 78 ILE 79
ALA 73 L$U 75 ASN 76 ASN 77 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 LEU 82
ALA 73 LEU 75 ASN 76 ASN 77 VAL 81
ALA 73 LEU 75 ASN 76 ASN 77 GLY 80
ALA 73 LEU 75 ASN 76 ASN 77 ILE 79
ALA 73 LEU 75 ASN 76 ASN 77 SER 78
ALA 73 ALA 74 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 GLY 80 LEU 82 GLY 83
ALA 73 ALA 74 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 GLY 80 VAL 81 LEU 82
ALA 73 ALA 74 ILE 79 LEU 82 GLY 83
ALA 73 ALA 74 ILE 79 VAL 8I GLY 83
ALA 73 ALA 74 ILE 79 VAL 81 LEU 82
ALA 73 ALA 74 ILE 79 GLY 80 GLY 83
ALA 73 ALA 74 ILE ?9 GLY 80 LEU 82
ALA 73 ALA 74 ILE 79 GLY 80 VAL 81
ALA 73 ALA 74 SER 78 LEU 82 GLY 83
ALA 73 ALA 74 SER 78 VAL 81 GLY 83
ALA 73 ALA 74 SER 78 VAL 81 LEU 82


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ALA 73 ALA ?4 SER 78 GLY 80 GLY 83
ALA 73 ALA 74 SER 78 GLY 80 LEU 82
ALA 73 ALA 74 SER 78 GLY 80 VAL 81
ALA 73 ALA 74 SER 78 ILE 79 GLY 83
ALA 73 ALA 74 SER 78 ILE 79 LEU 82
ALA 73 ALA 74 SER 78 ILE 79 VAL 81
ALA 73 ALA 74 SER 78 ILE 79 GLY 80
ALA 73 ALA 74 ASN 77 LEU 82 GLY 83
ALA 73 ALA 74 ASN 77 VAL 81 GLY 83
ALA 73 ALA 74 ASN 77 VAL 81 LEU 82
ALA 73 ALA 74 ASN ?7 GLY 80 GLY 83
ALA 73 ALA 74 ASN 77 GLY 80 LEU 82
ALA ?3 ALA 74 ASN 77 GLY 80 VAL 81
ALA 73 ALA 74 ASN 77 ILE 79 GLY 83
ALA 73 ALA 74 ASN 77 ILE 79 LEU 82
ALA 73 ALA 74 ASN 77 ILE 79 VAL 81
ALA 73 ALA 74 ASN 77 ILE ?9 GLY 80
ALA 73 ALA 74 ASN 77 SER 78 GLY 83
ALA 73 ALA 74 ASN 77 SER 78 LEU 82
ALA 73 ALA 74 ASN 77 SER 78 VAL 81
ALA 73 ALA 74 ASN 77 SER 78 GLY 80
ALA 73 ALA 74 ASN 77 SER 78 ILE 79
ALA 73 ALA 74 ASN 76 LEU 82 GLY 83
ALA 73 ALA 74 ASN 76 VAL 81 GLY 83
ALA 73 ALA 74 ASN 76 VAL 81 LEU 82
ALA 73 ALA 74 ASN 76 GLY 80 GLY 83
ALA 73 ALA 74 ASN 76 GLY 80 LEU 82
ALA 73 ALA 74 ASN 76 GLY 80 VAL 81
ALA 73 ALA 74 ASN 76 ILE 79 GLY 83
ALA 73 ALA 74 ASN 76 ILE 79 LEU 82
ALA 73 ALA 74 ASN 76 ILE 79 VAL 81
ALA 73 ALA 74 ASN 76 ILE 79 GLY 80
ALA 73 ALA 74 ASN ?6 SER 78 GLY 83
ALA 73 ALA 74 ASN 76 SER 78 LEU 82
ALA 73 ALA 74 ASN 76 SER 78 VAL 81
ALA 73 ALA 74 ASN 76 SER 78 GLY 80


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ALA 73 ALA 74 ASN 76 SER 78 ILE 79
ALA 73 ALA 74 ASN 76 ASN 77 GLY 83
ALA 73 ALA 74 ASN 76 ASN 77 LEU 82
ALA 73 ALA 74 ASN 76 ASN 77 VAL 81
ALA 73 ALA 74 ASN 76 ASN 77 GLY 80
ALA 73 ALA 74 ASN 76 ASN 77 ILE 79
ALA 73 ALA 74 ASN 76 ASN 77 SER 78
ALA 73 ALA 74 LEU 75 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 VAL 81 GLY 83
ALA 73 ALA 74 LEU 75 VAL 81 LEU 82
ALA 73 ALA ?4 LEU 75 GLY 80 GLY 83
ALA 73 ALA 74 LEU 75 GLY 80 LEU 82
ALA 73 ALA 74 LEU 75 GLY 80 VAL 81
ALA 73 ALA 74 LEU 75 ILE 79 GLY 83
ALA 73 ALA 74 LEU 75 ILE 79 LEU 82
ALA 73 ALA 74 LEU 75 ILE 79 VAL 81
ALA 73 ALA 74 LEU 75 ILE 79 GLY 80
ALA 73 ALA 74 LEU 75 SER 78 GLY 83
ALA 73 ALA 74 LEU 75 SER 78 LEU 82
ALA 73 ALA 74 LEU 75 SER 78 VAL 81
ALA 73 ALA 74 LEU 75 SER 78 GLY 80
ALA 73 ALA 74 LEU 75 SER 78 ILE 79
ALA 73 ALA 74 LEU 75 ASN 77 GLY 83
ALA 73 ALA 74 LEU 75 ASN 77 LEU 82
ALA 73 ALA 74 LEU 75 ASN 77 VAL 81
ALA 73 ALA 74 LEU 75 ASN 77 GLY 80
ALA 73 ALA 74 LEU 75 ASN 771LE 79
ALA 73 ALA 74 LEU 75 ASN 77 SER 78
ALA 73 ALA 74 LEU 75 ASN 76 GLY 83
ALA 73 ALA ?4 LEU 75 ASN 76 LEU 82
ALA 73 ALA 74 LEU 75 ASN 76 VAL 81
ALA 73 ALA 74 LEU 75 ASN 76 GLY 80
ALA 73 ALA 74 LEU 75 ASN 76 ILE 79
ALA 73 ALA 74 LEU 75 ASN 76 SER 78
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77


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Table 7
Sextuple Mutation Variants
SER 78 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ASN 77 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ASN 77 SER 78 GLY 80 VAL 81 LEU 82 GLY 83
ASN 77 SER 78 ILE 79 VAL 81 LEU 82 GLY 83
ASN 77 SER 78 ILE 79 GLY 80 LEU 82 GLY 83
ASN 77 SER 78 ILE 79 GLY 80 VAL 81 GLY 83
ASN 77 SER 78 ILE 79 GLY 80 VAL 81 LEU 82
ASN 76 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ASN 76 SER 78 GLY 80 VAL 81 LEU 82 GLY 83
ASN 76 SER 78 ILE 79 VAL 81 LEU 82 GLY 83
ASN 76 SER 78 ILE 79 GLY 80 LEU 82 GLY 83
ASN 76 SER 78 ILE 79 GLY 80 VAL 81 GLY 83
ASN 76 SER 78 ILE 79 GLY 80 VAL 81 LEU 82
ASN 76 ASN 77 GLY 80 VAL 81 LEU 82 GLY 83
ASN 76 ASN 77 ILE 79 VAL 81 LEU 82 GLY 83
ASN 76 ASN 77 ILE 79 GLY 80 LEU 82 GLY 83
ASN 76 ASN 77 ILE 79 GLY 80 VAL 81 GLY 83
ASN 76 ASN 77 ILE 79 GLY 80 VAL 81 LEU 82
ASN 76 ASN 77 SER 78 VAL 81 LEU 82 GLY 83
ASN 76 ASN 77 SER 78 GLY 80 LEU 82 GLY 83
ASN 76 ASN 77 SER 78 GLY 80 VAL 81 GLY 83
ASN 76 ASN 77 SER 78 GLY 80 VAL 81 LEU 82
ASN 76 ASN 77 SER 78 ILE 79 LEU 82 GLY 83
ASN 76 ASN 77 SER 78 ILE 79 VAL 81 GLY 83
ASN 76 ASN 77 SER 78 ILE 79 VAL 81 LEU 82
ASN 76 ASN 77 SER 78 ILE 79 GLY 80 GLY 83
ASN 76 ASN 77 SER 78 ILE 79 GLY 80 LEU 82
ASN 76 ASN 77 SER 78 ILE 79 GLY 80 VAL 81
LEU 75 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
LEU 75 SER 78 GLY 80 VAL 81 LEU 82 GLY 83
LEU 75 SER 78 ILE 79 VAL 81 LEU 82 GLY 83
LEU 75 SER 78 ILE 79 GLY 80 LEU 82 GLY 83
LEU 75 SER 78 ILE 79 GLY 80 VAL 81 GLY 83
LEU 75 SER 78 ILE 79 GLY 80 VAL 81 LEU 82


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LEU 75 ASN 77 GLY 80 VAL 81 LEU 82 GLY 83
LEU 75 ASN 77 ILE 79 VAL 81 LEU 82 GLY 83
LEU 75 ASN 77 ILE 79 GLY 80 LEU 82 GLY 83
LEU 75 ASN 77 ILE 79 GLY 80 VAL 81 GLY 83
LEU 75 ASN 77 ILE 79 GLY 80 VAL 81 LEU 82
LEU 75 ASN 77 SER 78 VAL 81 LEU 82 GLY 83
LEU 75 ASN 77 SER 78 GLY 80 LEU 82 GLY 83
LEU 75 ASN 77 SER 78 GLY 80 VAL 81 GLY 83
LEU 75 ASN 77 SER 78 GLY 80 VAL 81 LEU 82
LEU 75 ASN 77 SER 78 ILE 79 LEU 82 GLY 83
LEU 75 ASN 77 SER 78 ILE 79 VAL 81 GLY 83
LEU 75 ASN 77 SER 78 ILE 79 VAL 81 LEU 82
LEU 75 ASN 77 SER 78 ILE 79 GLY 80 GLY 83
LEU 75 ASN 77 SER 78 ILE 79 GLY 80 LEU 82
LEU 75 ASN 77 SER 78 ILE 79 GLY 80 VAL 81
LEU 75 ASN 76 GLY 80 VAL 81 LEU 82 GLY 83
LEU 75 ASN 76 ILE 79 VAL 81 LEU 82 GLY 83
LEU 75 ASN 76 ILE 79 GLY 80 LEU 82 GLY 83
LEU 75 ASN 76 ILE 79 GLY . 80 VAL 81 GLY 83
LEU 75 ASN 76 ILE 79 GLY 80 VAL 81 LEU 82
LEU 75 ASN 76 SER 78 VAL 81 LEU 82 GLY 83
LEU 75 ASN 76 SER 78 GLY 80 LEU 82 GLY 83
LEU 75 ASN 76 SER 78 GLY 80 VAL 81 GLY 83
LEU 75 ASN 76 SER 78 GLY 80 VAL 81 LEU 82
LEU 75 ASN 76 SER 78 ILE 79 LEU 82 GLY 83
LEU 75 ASN 76 SER 78 ILE 79 VAL 81 GLY 83
LEU 75 ASN 76 SER 78 ILE 79 VAL 81 LEU 82
LEU 75 ASN 76 SER 78 ILE 79 GLY 80 GLY 83
LEU 75 ASN 76 SER 78 ILE 79 GLY 80 LEU 82
LEU 75 ASN 76 SER 78 ILE 79 GLY 80 VAL 81
LEU 75 ASN 76 ASN 77 VAL 81 LEU 82 GLY 83
LEU 75 ASN 76 ASN 77 GLY 80 LEU 82 GLY 83
LEU 75 ASN 76 ASN 77 GLY 80 VAL 81 GLY 83
LEU 75 ASN 76 ASN 77 GLY 80 VAL 81 LEU 82
LEU 75 ASN 76 ASN 77 ILE 79 LEU 82 GLY 83
LEU 75 ASN 76 ASN 77 ILE 79 VAL 81 GLY 83


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LEU 75 ASN 76 ASN 77 ILE 79 VAL 81 LEU 82
LEU 75 ASN 76 ASN 77 ILE 79 GLY 80 GLY 83
LEU 75 ASN 76 ASN 77 ILE 79 GLY 80 LEU 82
LEU 75 ASN 76 ASN 77 ILE 79 GLY 80 VAL 81
LEU 75 ASN 76 ASN ?7 SER 78 LEU 82 GLY 83
LEU 75 ASN 76 ASN 77 SER 78 VAL 81 GLY 83
LEU 75 ASN 76 ASN 77 SER 78 VAL 81 LEU 82
LEU 75 ASN 76 ASN 77 SER 78 GLY 80 GLY 83
LEU 75 ASN 76 ASN 77 SER 78 GLY 80 LEU 82
LEU 75 ASN 76 ASN 77 SER 78 GLY 80 VAL 81
LEU 75 ASN 76 ASN 77 SER 78 ILE 79 GLY 83
LEU 75 ASN 76 ASN 77 SER 78 ILE 79 LEU 82
LEU 75 ASN 76 ASN ?7 SER 78 ILE 79 VAL 81
LEU 75 ASN 76 ASN 77 SER 78 ILE 79 GLY 80
ALA 74 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 74 SER 78 GLY 80 VAL 81 LEU 82 GLY 83
ALA 74 SER 78 ILE 79 VAL 81 LEU 82 GLY 83
ALA 74 SER 78 ILE 79 GLY 80 LEU 82 GLY 83
ALA 74 SER 78 ILE 79 GLY 80 VAL 81 GLY 83
ALA 74 SER 78 ILE 79 GLY 80 VAL 81 LEU 82
ALA ?4 ASN 77 GLY 80 VAL 81 LEU 82 GLY 83
ALA 74 ASN 77 ILE 79 VAL 81 LEU 82 GLY 83
ALA 74 ASN 77 ILE 79 GLY 80 LEU 82 GLY 83
ALA ?4 ASN 77 ILE 79 GLY 80 VAL 81 GLY 83
ALA 74 ASN 77 ILE 79 GLY 80 VAL 81 LEU 82
ALA 74 ASN 77 SER 78 VAL 81 LEU 82 GLY 83
ALA 74 ASN 77 SER 78 GLY 80 LEU 82 GLY 83
ALA 74 ASN 77 SER 78 GLY 80 VAL 81 GLY 83
ALA 74 ASN 77 SER 78 GLY 80 VAL 81 LEU 82
ALA 74 ASN 77 SER 78 ILE 79 LEU 82 GLY 83
ALA 74 ASN 77 SER 78 ILE 79 VAL 81 GLY 83
ALA 74 ASN 77 SER 78 ILE 79 VAL 81 LEU 82
ALA 74 ASN 77 SER 78 ILE 79 GLY 80 GLY 83
ALA 74 ASN 77 SER 78 ILE 79 GLY 80 LEU 82
ALA 74 ASN 77 SER 78 ILE 79 GLY 80 VAL 81
ALA 74 ASN 76 GLY 80 VAL 81 LEU 82 GLY 83


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ALA 74 ASN 76 ILE 79 VAL 81 LEU 82 GLY 83
ALA 74 ASN 76 ILE 79 GLY 80 LEU 82 GLY 83
ALA 74 ASN 761LE 79 GLY 80 VAL 81 GLY 83
ALA 74 ASN 76 ILE 79 GLY 80 VAL 81 LEU 82
ALA 74 ASN 76 SER 78 VAL 81 LEU 82 GLY 83
ALA 74 ASN 76 SER ?8 GLY 80 LEU 82 GLY 83
ALA 74 ASN 76 SER 78 GLY 80 VAL 81 GLY 83
ALA 74 ASN 76 SER 78 GLY 80 VAL 81 LEU 82
ALA 74 ASN 76 SER 78 ILE 79 LEU 82 GLY 83
ALA 74 ASN 76 SER 78 ILE 79 VAL 81 GLY 83
ALA ?4 ASN 76 SER 78 ILE 79 VAL 81 LEU 82
ALA 74 ASN 76 SER 78 ILE 79 GLY 80 GLY 83
ALA 74 ASN 76 SER 78 ILE 79 GLY 80 LEU 82
ALA 74 ASN 76 SER 78 ILE 79 GLY 80 VAL 81
ALA 74 ASN 76 ASN 77 VAL 81 LEU 82 GLY 83
ALA 74 ASN 76 ASN 77 GLY 80 LEU 82 GLY 83
ALA 74 ASN 76 ASN 77 GLY 80 VAL 81 GLY 83
ALA 74 ASN 76 ASN 77 GLY 80 VAL 81 LEU 82
ALA 74 ASN 76 ASN 77 ILE 79 LEU 82 GLY 83
ALA 74 ASN 76 ASN 77 ILE 79 VAL 81 GLY 83
ALA 74 ASN 76 ASN 77 ILE 79 VAL 81 LEU 82
ALA 74 ASN 76 ASN 77 ILE 79 GLY 80 GLY 83
ALA 74 ASN 76 ASN 77 ILE 79 GLY 80 LEU 82
ALA 74 ASN 76 ASN 77 ILE 79 GLY 80 VAL 81
ALA 74 ASN 76 ASN 77 SER 78 LEU 82 GLY 83
ALA 74 ASN 76 ASN 77 SER 78 VAL 81 GLY 83
ALA 74 ASN 76 ASN 77 SER 78 VAL 81 LEU 82
ALA 74 ASN 76 ASN 77 SER 78 GLY 80 GLY 83
ALA ?4 ASN 76 ASN 77 SER 78 GLY 80 LEU 82
ALA 74 ASN 76 ASN ?7 SER 78 GLY 80 VAL 81
ALA 74 ASN 76 ASN 77 SER 78 ILE 79 GLY 83
ALA 74 ASN 76 ASN 77 SER 78 ILE 79 LEU 82
ALA 74 ASN 76 ASN 77 SER 78 ILE 79 VAL 81
ALA 74 ASN 76 ASN 77 SER 78 ILE 79 GLY 80
ALA 74 LEU 75 GLY 80 VAL 81 LEU 82 GLY 83
ALA 74 LEU 75 ILE 79 VAL 81 LEU 82 GLY 83


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ALA 74 LEU 75 ILE 79 GLY 80 LEU 82 GLY 83
ALA 74 LEU 75 ILE 79 GLY 80 VAL 81 GLY 83
ALA 74 LEU 75 ILE 79 GLY 80 VAL 81 LEU 82
ALA 74 LEU 75 SER 78 VAL 81 LEU 82 GLY 83
ALA 74 LEU 75 SER 78 GLY 80 LEU 82 GLY 83
ALA 74 LEU 75 SER 78 GLY 80 VAL 81 GLY 83
ALA 74 LEU 75 SER 78 GLY 80 VAL 81 LEU 82
ALA 74 LEU 75 SER 78 ILE 79 LEU 82 GLY 83
ALA 74 LEU 75 SER 78 ILE 79 VAL 81 GLY 83
ALA 74 LEU 75 SER 78 ILE 79 VAL 81 LEU 82
ALA 74 LEU 75 SER 78 ILE 79 GLY 80 GLY 83
ALA 74 LEU 75 SER 78 ILE 79 GLY 80 LEU 82
ALA 74 LEU 75 SER 78 ILE 79 GLY 80 VAL 81
ALA 74 LEU 75 ASN 77 VAL 81 LEU 82 GLY 83
ALA 74 LEU 75 ASN 77 GLY 80 LEU 82 GLY 83
ALA 74 LEU 75 ASN 77 GLY 80 VAL 81 GLY 83
ALA 74 LEU 75 ASN 77 GLY 80 VAL 81 LEU 82
ALA 74 LEU 75 ASN 77 ILE 79 LEU 82 GLY 83
ALA 74 LEU 75 ASN 77 ILE 79 VAL 81 GLY 83
ALA 74 LEU 75 ASN 77 ILE 79 VAL 81 LEU 82
ALA 74 LEU 75 ASN 77 ILE 79 GLY 80 GLY 83
ALA 74 LEU 75 ASN 77 ILE 79 GLY 80 LEU 82
ALA 74 LEU 75 ASN 77 ILE 79 GLY 80 VAL 81
ALA 74 LEU 75 ASN 77 SER 78 LEU 82 GLY 83
ALA 74 LEU 75 ASN 77 SER 78 VAL 81 GLY 83
ALA 74 LEU 75 ASN 77 SER 78 VAL 81 LEU 82
ALA 74 LEU 75 ASN 77 SER 78 GLY 80 GLY 83
ALA 74 LEU 75 ASN 77 SER 78 GLY 80 LEU 82
ALA 74 LEU 75 ASN 77 SER 78 GLY 80 VAL 81
ALA 74 LEU 75 ASN 77 SER 78 ILE 79 GLY 83
ALA 74 LEU 75 ASN 77 SER 78 ILE 79 LEU 82
ALA 74 LEU 75 ASN 77 SER 78 ILE 79 VAL 81
ALA 74 LEU 75 ASN 77 SER 78 ILE 79 GLY 80
ALA 74 LEU 75 ASN 76 VAL 81 LEU 82 GLY 83
ALA 74 LEU 75 ASN 76 GLY 80 LEU 82 GLY 83
ALA 74 LEU 75 ASN 76 GLY 80 VAL 81 GLY 83


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ALA 74 LEU 75 ASN 76 GLY 80 VAL 81 LEU 82
ALA 74 LEU 75 ASN ?6 ILE 79 LEU 82 GLY 83
ALA 74 LEU 75 ASN ?6 ILE 79 VAL 81 GLY 83
ALA 74 LEU 75 ASN 76 ILE 79 VAL 81 LEU 82
ALA 74 LEU 75 ASN 76 ILE 79 GLY 80 GLY 83
ALA 74 LEU 75 ASN ?6 ILE 79 GLY 80 LEU 82
ALA 74 LEU 75 ASN 76 ILE 79 GLY 80 VAL 81
ALA 74 LEU 75 ASN ?6 SER 78 LEU 82 GLY 83
ALA 74 LEU 75 ASN 76 SER 78 VAL 81 GLY 83
ALA 74 LEU 75 ASN 76 SER 78 VAL 81 LEU 82
ALA 74 LEU 75 ASN 76 SER 78 GLY 80 GLY 83
ALA 74 LEU 75 ASN 76 SER 78 GLY 80 LEU 82
ALA 74 LEU 75 ASN 76 SER 78 GLY 80 VAL 81
ALA 74 LEU 75 ASN 76 SER 78 ILE 79 GLY 83
ALA 74 LEU ?5 ASN ?6 SER 78 ILE 79 LEU 82
ALA 74 LEU 75 ASN ?6 SER 78 ILE 79 VAL 81
ALA 74 LEU 75 ASN 76 SER 78 ILE 79 GLY 80
ALA 74 LEU 75 ASN ?6 ASN 77 LEU 82 GLY 83
ALA 74 LEU 75 ASN 76 ASN 77 VAL 81 GLY 83
ALA ?4 LEU 75 ASN 76 ASN 77 VAL 81 LEU 82
ALA 74 LEU 75 ASN 76 ASN 77 GLY 80 GLY 83
ALA 74 LEU 75 ASN 76 ASN 77 GLY 80 LEU 82
ALA 74 LEU 75 ASN 76 ASN 77 GLY 80 VAL 81
ALA 74 LEU ?5 ASN 76 ASN ?7 ILE 79 GLY 83
ALA 74 LEU 75 ASN 76 ASN 77 ILE 79 LEU 82
ALA 74 LEU 75 ASN 76 ASN ?? ILE 79 VAL 81
ALA 74 LEU 75 ASN 76 ASN ?7 ILE 79 GLY 80
ALA 74 LEU 75 ASN 76 ASN 77 SER ?8 GLY 83
ALA 74 LEU 75 ASN 76 ASN 77 SER 78 LEU 82
ALA 74 LEU 75 ASN 76 ASN 7? SER 78 VAL 81
ALA 74 LEU 75 ASN 76 ASN 77 SER 78 GLY 80
ALA 74 LEU 75 ASN ?6 ASN 77 SER 78 ILE 79
ALA 73 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 SER 78 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 SER 78 ILE 79 VAL 81 LEU 82 GLY 83
ALA 73 SER 78 ILE 79 GLY 80 LEU 82 GLY 83


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ALA 73 SER 78 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 SER 78 ILE 79 GLY 80 VAL 81 LEU 82
ALA 73 ASN 77 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ASN ?7 ILE 79 VAL 81 LEU 82 GLY 83
ALA 73 ASN 77 ILE 79 GLY 80 LEU 82 GLY 83
ALA ?3 ASN 77 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 ASN 77 ILE 79 GLY 80 VAL 81 LEU 82
ALA 73 ASN 77 SER 78 VAL 81 LEU 82 GLY 83
ALA 73 ASN 77 SER 78 GLY 80 LEU 82 GLY 83
ALA 73 ASN 77 SER 78 GLY 80 VAL 81 GLY 83
ALA 73 ASN 77 SER 78 GLY 80 VAL 81 LEU 82
ALA 73 ASN 77 SER 78 ILE 79 LEU 82 GLY 83
ALA 73 ASN 77 SER 78 ILE 79 VAL 81 GLY 83
ALA 73 ASN 77 SER 78 ILE 79 VAL 81 LEU 82
ALA 73 ASN 77 SER 78 ILE 79 GLY 80 GLY 83
ALA 73 ASN 77 SER 78 ILE 79 GLY 80 LEU 82
ALA 73 ASN 77 SER 78 ILE 79 GLY 80 VAL 81
ALA 73 ASN 76 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ASN 76 ILE 79 VAL 81 LEU 82 GLY 83
ALA 73 ASN 76 ILE 79 GLY 80 LEU 82 GLY 83
ALA 73 ASN 76 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 ASN 76 ILE 79 GLY 80 VAL 81 LEU 82
ALA 73 ASN 76 SER 78 VAL 81 LEU 82 GLY 83
ALA 73 ASN 76 SER 78 GLY 80 LEU 82 GLY 83
ALA 73 ASN 76 SER 78 GLY 80 VAL 81 GLY 83
ALA 73 ASN 76 SER 78 GLY 80 VAL 81 LEU 82
ALA 73 ASN 76 SER 78 ILE 79 LEU 82 GLY 83
ALA 73 ASN 76 SER 78 ILE 79 VAL 81 GLY 83
ALA 73 ASN 76 SER 78 ILE 79 VAL 81 LEU 82
ALA 73 ASN 76 SER 78 ILE 79 GLY 80 GLY 83
ALA 73 ASN 76 SER 78 ILE 79 GLY 80 LEU 82
ALA 73 ASN 76 SER 78 ILE 79 GLY 80 VAL 81
ALA 73 ASN 76 ASN 77 VAL 81 LEU 82 GLY 83
ALA 73 ASN 76 ASN 77 GLY 80 LEU 82 GLY 83
ALA 73 ASN 76 ASN 77 GLY 80 VAL 81 GLY 83
ALA 73 ASN 76 ASN 77 GLY 80 VAL 81 LEU 82


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ALA 73 ASN 76 ASN 77 ILE 79 LEU 82 GLY 83
ALA 73 ASN 76 ASN 77 ILE 79 VAL 81 GLY 83
ALA 73 ASN 76 ASN 77 ILE 79 VAL 81 LEU 82
ALA 73 ASN 76 ASN 77 ILE 79 GLY 80 GLY 83
ALA 73 ASN 76 ASN 77 ILE 79 GLY 80 LEU 82
ALA 73 ASN 76 ASN 77 ILE 79 GLY 80 VAL 81
ALA 73 ASN 76 ASN 77 SER 78 LEU 82 GLY 83
ALA 73 ASN 76 ASN 77 SER 78 VAL 81 GLY 83
ALA 73 ASN 76 ASN 77 SER 78 VAL 81 LEU 82
ALA 73 ASN 76 ASN 77 SER 78 GLY 80 GLY 83
ALA 73 ASN 76 ASN 77 SER 78 GLY 80 LEU 82
ALA 73 ASN 76 ASN 77 SER 78 GLY 80 VAL 81
ALA 73 ASN 76 ASN 77 SER 78 ILE 79 GLY 83
ALA 73 ASN 76 ASN 77 SER 78 ILE 79 LEU 82
ALA 73 ASN 76 ASN 77 SER 78 ILE 79 VAL 81
ALA 73 ASN 76 ASN 77 SER 78 ILE 79 GLY 80
ALA 73 LEU 75 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 LEU 75 ILE 79 VAL 81 LEU 82 GLY 83
ALA 73 LEU 75 ILE 79 GLY 80 LEU 82 GLY 83
ALA 73 LEU 75 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 LEU 75 ILE 79 GLY 80 VAL 81 LEU 82
ALA 73 LEU 75 SER 78 VAL 81 LEU 82 GLY 83
ALA 73 LEU 75 SER 78 GLY 80 LEU 82 GLY 83
ALA 73 LEU 75 SER 78 GLY 80 VAL 81 GLY 83
ALA 73 LEU 75 SER 78 GLY 80 VAL 81 LEU 82
ALA 73 LEU 75 SER 78 ILE 79 LEU 82 GLY 83
ALA 73 LEU 75 SER 78 ILE 79 VAL 81 GLY 83
ALA 73 LEU 75 SER 78 ILE 79 VAL 81 LEU 82
ALA 73 LEU 75 SER 78 ILE 79 GLY 80 GLY 83
ALA 73 LEU 75 SER 78 ILE 79 GLY 80 LEU 82
ALA 73 LEU 75 SER 78 ILE 79 GLY 80 VAL 81
ALA 73 LEU 75 ASN 77 VAL 81 LEU 82 GLY 83
ALA 73 LEU 75 ASN 77 GLY 80 LEU 82 GLY 83
ALA 73 LEU 75 ASN 77 GLY 80 VAL 81 GLY 83
ALA 73 LEU 75 ASN 77 GLY 80 VAL 81 LEU 82
ALA 73 LEU 75 ASN 77 ILE 79 LEU 82 GLY 83


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ALA 73 LEU 75 ASN 77 ILE 79 VAL 81 GLY 83
ALA 73 LEU 75 ASN 77 ILE 79 VAL 81 LEU 82
ALA 73 LEU 75 ASN 77 ILE 79 GLY 80 GLY 83
ALA 73 LEU 75 ASN 77 ILE 79 GLY 80 LEU 82
ALA 73 LEU 75 ASN 77 ILE 79 GLY 80 VAL 81
ALA 73 LEU 75 ASN 77 SER 78 LEU 82 GLY 83
ALA 73 LEU 75 ASN 77 SER 78 VAL 81 GLY 83
ALA 73 LEU 75 ASN 77 SER 78 VAL 81 LEU 82
ALA 73 LEU 75 ASN 77 SER 78 GLY 80 GLY 83
ALA 73 LEU 75 ASN 77 SER 78 GLY 80 LEU 82
ALA 73 LEU 75 ASN 77 SER 78 GLY 80 VAL 81
ALA 73 LEU 75 ASN 77 SER 78 ILE 79 GLY 83
ALA 73 LEU 75 ASN 77 SER 78 ILE 79 LEU 82
ALA 73 LEU 75 ASN 77 SER 78 ILE 79 VAL 81
ALA 73 LEU 75 ASN 77 SER 78 ILE 79 GLY 80
ALA 73 LEU 75 ASN 76 VAL 81 LEU 82 GLY 83
ALA 73 LEU 75 ASN 76 GLY 80 LEU 82 GLY 83
ALA 73 LEU 75 ASN 76 GLY 80 VAL 81 GLY 83
ALA 73 LEU 75 ASN 76 GLY 80 VAL 81 LEU 82
ALA 73 LEU 75 ASN 76 ILE ?9 LEU 82 GLY 83
ALA 73 LEU 75 ASN 76 ILE 79 VAL 81 GLY 83
ALA 73 LEU 75 ASN 76 ILE 79 VAL 81 LEU 82
ALA 73 LEU 75 ASN 76 ILE 79 GLY 80 GLY 83
ALA 73 LEU 75 ASN 76 ILE 79 GLY 80 LEU 82
ALA 73 LEU 75 ASN 76 ILE 79 GLY 80 VAL 81
ALA 73 LEU 75 ASN 76 SER 78 LEU 82 GLY 83
ALA 73 LEU 75 ASN 76 SER 78 VAL 81 GLY 83
ALA 73 LEU 75 ASN 76 SER 78 VAL 81 LEU 82
ALA 73 LEU 75 ASN 76 SER 78 GLY 80 GLY 83
ALA 73 LEU 75 ASN 76 SER 78 GLY 80 LEU 82
ALA 73 LEU 75 ASN 76 SER 78 GLY 80 VAL 81
ALA 73 LEU 75 ASN 76 SER 78 ILE 79 GLY 83
ALA 73 LEU 75 ASN 76 SER 78 ILE 79 LEU 82
ALA 73 LEU 75 ASN 76 SER 78 ILE 79 VAL 81
ALA 73 LEU 75 ASN 76 SER 78 ILE 79 GLY 80
ALA 73 LEU 75 ASN 76 ASN 77 LEU 82 GLY 83


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ALA 73 LEU 75 ASN 76 ASN 77 VAL 81 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 VAL 81 LEU 82
ALA 73 LEU 75 ASN 76 ASN 77 GLY 80 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 GLY 80 LEU 82
ALA 73 LEU 75 ASN 76 ASN 77 GLY 80 VAL 81
ALA 73 LEU 75 ASN 76 ASN 77 ILE 79 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 ILE 79 LEU 82
ALA 73 LEU 75 ASN 76 ASN 7? ILE 79 VAL 81
ALA 73 LEU 75 ASN 76 ASN 77 ILE 79 GLY 80
ALA 73 LEU 75 ASN 76 ASN 77 SER 78 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 SER 78 LEU 82
ALA 73 LEU 75 ASN 76 ASN 77 SER 78 VAL 81
ALA 73 LEU 75 ASN 76 ASN 77 SER 78 GLY 80
ALA 73 LEU 75 ASN 76 ASN 77 SER 78 ILE 79
ALA 73 ALA ?4 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 ILE 79 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 ILE 79 GLY 80 LEU 82 GLY 83
ALA 73 ALA 74 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 ILE 79 GLY 80 VAL 81 LEU 82
ALA 73 ALA 74 SER 78 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 SER 78 GLY 80 LEU 82 GLY 83
ALA 73 ALA 74 SER 78 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 SER 78 GLY 80 VAL 81 LEU 82
ALA 73 ALA 74 SER 78 ILE 79 LEU 82 GLY 83
ALA 73 ALA 74 SER 78 ILE 79 VAL 81 GLY 83
ALA 73 ALA 74 SER 78 ILE 79 VAL 81 LEU 82
ALA 73 ALA 74 SER 78 ILE 79 GLY 80 GLY 83
ALA 73 ALA 74 SER 78 ILE 79 GLY 80 LEU 82
ALA 73 ALA 74 SER 78 ILE 79 GLY 80 VAL 81
ALA 73 ALA 74 ASN 77 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 ASN 77 GLY 80 LEU 82 GLY 83
ALA 73 ALA 74 ASN 77 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 ASN 77 GLY 80 VAL 81 LEU 82
ALA 73 ALA 74 ASN 77 ILE 79 LEU 82 GLY 83
ALA 73 ALA 74 ASN 77 ILE 79 VAL 81 GLY 83
ALA 73 ALA 74 ASN 77 ILE 79 VAL 81 LEU 82


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ALA 73 ALA 74 ASN 77 ILE 79 GLY 80 GLY 83
ALA 73 ALA 74 ASN 77 ILE 79 GLY 80 LEU 82
ALA 73 ALA 74 ASN 77 ILE 79 GLY 80 VAL 81
ALA 73 ALA 74 ASN 77 SER 78 LEU 82 GLY 83
ALA 73 ALA 74 ASN 77 SER 78 VAL 81 GLY 83
ALA 73 ALA 74 ASN 77 SER 78 VAL 81 LEU 82
ALA 73 ALA 74 ASN 77 SER 78 GLY 80 GLY 83
ALA 73 ALA 74 ASN 77 SER 78 GLY 80 LEU 82
ALA 73 ALA 74 ASN 77 SER 78 GLY 80 VAL 81
ALA 73 ALA 74 ASN 77 SER 78 ILE 79 GLY 83
ALA 73 ALA 74 ASN 77 SER 78 ILE 79 LEU 82
ALA 73 ALA 74 ASN 77 SER 78 ILE 79 VAL 81
ALA 73 ALA 74 ASN 77 SER 78 ILE 79 GLY 80
ALA 73 ALA 74 ASN 76 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 ASN 76 GLY 80 LEU 82 GLY 83
ALA 73 ALA 74 ASN 76 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 ASN 76 GLY 80 VAL 81 LEU 82
ALA 73 ALA 74 ASN 76 ILE 79 LEU 82 GLY 83
ALA 73 ALA 74 ASN 76 ILE 79 VAL 81 GLY 83
ALA 73 ALA 74 ASN 76 ILE 79 VAL 81 LEU 82
ALA 73 ALA 74 ASN 76 ILE 79 GLY 80 GLY 83
ALA 73 ALA 74 ASN 76 ILE 79 GLY 80 LEU 82
ALA 73 ALA 74 ASN 76 ILE 79 GLY 80 VAL 81
ALA 73 ALA 74 ASN 76 SER 78 LEU 82 GLY 83
ALA 73 ALA 74 ASN 76 SER 78 VAL 81 GLY 83
ALA 73 ALA 74 ASN 76 SER 78 VAL 81 LEU 82
ALA 73 ALA 74 ASN ?6 SER 78 GLY 80 GLY 83
ALA 73 ALA 74 ASN 76 SER 78 GLY 80 LEU 82
ALA 73 ALA 74 ASN 76 SER 78 GLY 80 VAL 81
ALA 73 ALA 74 ASN 76 SER 78 ILE 79 GLY 83
ALA 73 ALA 74 ASN 76 SER 78 ILE 79 LEU 82
ALA 73 ALA 74 ASN 76 SER 78 ILE 79 VAL 81
ALA 73 ALA 74 ASN 76 SER 78 ILE 79 GLY 80
ALA 73 ALA 74 ASN 76 ASN 77 LEU 82 GLY 83
ALA 73 ALA 74 ASN 76 ASN 77 VAL 81 GLY 83
ALA 73 ALA 74 ASN 76 ASN 77 VAL 81 LEU 82


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ALA 73 ALA 74 ASN 76 ASN 77 GLY 80 GLY 83
ALA 73 ALA 74 ASN 76 ASN 77 GLY 80 LEU 82
ALA 73 ALA 74 ASN 76 ASN 77 GLY 80 VAL 81
ALA 73 ALA 74 ASN ?6 ASN 77 ILE 79 GLY 83
ALA 73 ALA 74 ASN 76 ASN 77 ILE 79 LEU 82
ALA 73 ALA 74 ASN 76 ASN 77 ILE 79 VAL 81
ALA 73 ALA 74 ASN 76 ASN 77 ILE 79 GLY 80
ALA 73 ALA 74 ASN 76 ASN 77 SER 78 GLY 83
ALA 73 ALA 74 ASN 76 ASN 77 SER 78 LEU 82
ALA 73 ALA 74 ASN 76 ASN 77 SER 78 VAL 81
ALA 73 ALA 74 ASN 76 ASN 77 SER 78 GLY 80
ALA 73 ALA 74 ASN 76 ASN 77 SER 78 ILE 79
ALA 73 ALA 74 LEU 75 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 GLY 80 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 LEU 75 GLY 80 VAL 81 LEU 82
ALA 73 ALA 74 LEU 75 ILE 79 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ILE 79 VAL 81 GLY 83
ALA 73 ALA 74 LEU 75 ILE 79 VAL 81 LEU 82
ALA 73 ALA 74 LEU 75 ILE 79 GLY 80 GLY 83
ALA 73 ALA 74 LEU 75 ILE 79 GLY 80 LEU 82
ALA 73 ALA 74 LEU 75 ILE 79 GLY 80 VAL 81
ALA 73 ALA 74 LEU 75 SER 78 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 SER 78 VAL 81 GLY 83
ALA 73 ALA 74 LEU 75 SER 78 VAL 81 LEU 82
ALA 73 ALA 74 LEU 75 SER 78 GLY 80 GLY 83
ALA 73 ALA 74 LEU 75 SER 78 GLY 80 LEU 82
ALA 73 ALA 74 LEU 75 SER 78 GLY 80 VAL 81
ALA 73 ALA 74 LEU 75 SER 78 ILE 79 GLY 83
ALA 73 ALA 74 LEU 75 SER 78 ILE 79 LEU 82
ALA 73 ALA 74 LEU 75 SER 78 ILE 79 VAL 81
ALA 73 ALA 74 LEU 75 SER 78 ILE 79 GLY 80
ALA 73 ALA 74 LEU 75 ASN 77 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 77 VAL 81 GLY 83
ALA 73 ALA 74 LEU 75 ASN 77 VAL 81 LEU 82
ALA 73 ALA 74 LEU 75 ASN 77 GLY 80 GLY 83


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ALA 73 ALA 74 LEU 75 ASN 77 GLY 80 LEU 82
ALA 73 ALA 74 LEU 75 ASN 77 GLY 80 VAL 81
ALA 73 ALA 74 LEU 75 ASN 77 ILE 79 GLY 83
ALA 73 ALA 74 LEU 75 ASN 77 ILE 79 LEU 82
ALA 73 ALA 74 LEU 75 ASN 77 ILE 79 VAL 81
ALA 73 ALA 74 LEU 75 ASN 77 ILE 79 GLY 80
ALA 73 ALA 74 LEU 75 ASN 77 SER 78 GLY 83
ALA 73 ALA 74 LEU 75 ASN 77 SER 78 LEU 82
ALA 73 ALA 74 LEU 75 ASN 77 SER 78 VAL 81
ALA 73 ALA 74 LEU 75 ASN 77 SER 78 GLY 80
ALA 73 ALA 74 LEU 75 ASN 77 SER 78 ILE 79
ALA 73 ALA 74 LEU 75 ASN 76 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 VAL 81 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 VAL 81 LEU 82
ALA 73 ALA 74 LEU 75 ASN 76 GLY 80 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 GLY 80 LEU 82
ALA 73 ALA 74 LEU 75 ASN 76 GLY 80 VAL 81
ALA 73 ALA 74 LEU 75 ASN 76 ILE 79 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ILE 79 LEU 82
ALA 73 ALA 74 LEU 75 ASN 76 ILE 79 VAL 81
ALA 73 ALA 74 LEU 75 ASN 76 ILE 79 GLY 80
ALA 73 ALA 74 LEU 75 ASN 76 SER 78 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 SER 78 LEU 82
ALA 73 ALA 74 LEU 75 ASN 76 SER 78 VAL 81
ALA 73 ALA 74 LEU 75 ASN 76 SER 78 GLY 80
ALA 73 ALA 74 LEU 75 ASN 76 SER ?8 ILE 79
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 LEU 82
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 VAL 81
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 GLY 80
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 ILE 79
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 SER 78
Table 8
Septuple Mutation Variants
ASN 77 SER 78 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83


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ASN 76 SER 78 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ASN 76 ASN 771LE 79 GLY 80 VAL 81 LEU 82 GLY 83
ASN ?6 ASN 77 SER 78 GLY 80 VAL 81 LEU 82 GLY 83
ASN 76 ASN 77 SER 78 ILE 79 VAL 81 LEU 82 GLY 83
ASN 76 ASN 77 SER 78 ILE 79 GLY 80 LEU 82 GLY 83
ASN 76 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 GLY 83
ASN 76 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 LEU 82
LEU 75 SER 78 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
LEU . 75 ASN 77 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
LEU 75 ASN 77 SER 78 GLY 80 VAL 81 LEU 82 GLY 83
LEU 75 ASN 77 SER 78 ILE 79 VAL 81 LEU 82 GLY 83
LEU 75 ASN 77 SER 78 ILE 79 GLY 80 LEU 82 GLY 83
LEU 75 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 GLY 83
LEU 75 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 LEU 82
LEU 75 ASN 761LE 79 GLY 80 VAL 81 LEU 82 GLY 83
LEU 75 ASN 76 SER 78 GLY 80 VAL 81 LEU 82 GLY 83
LEU 75 ASN 76 SER 78 ILE 79 VAL 81 LEU 82 GLY 83
LEU 75 ASN 76 SER 78 ILE 79 GLY 80 LEU 82 GLY 83
LEU 75 ASN 76 SER 78 ILE 79 GLY 80 VAL 81 GLY 83
LEU 75 ASN 76 SER 78 ILE 79 GLY 80 VAL 81 LEU 82
LEU 75 ASN 76 ASN 77 GLY 80 VAL 81 LEU 82 GLY 83
LEU 75 ASN 76 ASN 77 ILE 79 VAL 81 LEU 82 GLY 83
LEU 75 ASN 76 ASN 77 ILE 79 GLY 80 LEU 82 GLY 83
LEU 75 ASN 76 ASN 77 ILE 79 GLY 80 VAL 81 GLY 83
LEU 75 ASN 76 ASN 77 ILE 79 GLY 80 VAL 81 LEU 82
LEU 75 ASN 76 ASN 77 SER 78 VAL 81 LEU 82 GLY 83
LEU 75 ASN 76 ASN 77 SER 78 GLY 80 LEU 82 GLY 83
LEU 75 ASN 76 ASN 77 SER 78 GLY 80 VAL 81 GLY 83
LEU 75 ASN 76 ASN 77 SER 78 GLY 80 VAL 81 LEU 82
LEU 75 ASN 76 ASN 77 SER 78 ILE 79 LEU 82 GLY 83
LEU 75 ASN 76 ASN 77 SER 78 ILE 79 VAL 81 GLY 83
LEU 75 ASN 76 ASN 77 SER 78 ILE 79 VAL 81 LEU 82
LEU 75 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 GLY 83
LEU 75 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 LEU 82
LEU 75 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 VAL 81
ALA 74 SER 78 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83


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ALA 74 ASN 77 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 74 ASN 77 SER 78 GLY 80 VAL 81 LEU 82 GLY 83
ALA 74 ASN 77 SER 78 ILE 79 VAL 81 LEU 82 GLY 83
ALA 74 ASN 77 SER 78 ILE 79 GLY 80 LEU 82 GLY 83
ALA 74 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 GLY 83
ALA 74 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 LEU 82
ALA 74 ASN 76 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 74 ASN 76 SER 78 GLY 80 VAL 81 LEU 82 GLY 83
ALA 74 ASN 76 SER 78 ILE 79 VAL 81 LEU 82 GLY 83
ALA 74 ASN 76 SER 78 ILE 79 GLY 80 LEU 82 GLY 83
ALA 74 ASN 76 SER 78 ILE 79 GLY 80 VAL 81 GLY 83
ALA 74 ASN 76 SER 78 ILE 79 GLY 80 VAL 81 LEU 82
ALA 74 ASN 76 ASN 77 GLY 80 VAL 81 LEU 82 GLY 83
ALA ?4 ASN 76 ASN 77 ILE 79 VAL 81 LEU 82 GLY 83
ALA 74 ASN 76 ASN 77 ILE 79 GLY 80 LEU 82 GLY 83
ALA 74 ASN 76 ASN 77 ILE 79 GLY 80 VAL 81 GLY 83
ALA 74 ASN 76 ASN 77 ILE 79 GLY 80 VAL 81 LEU 82
ALA 74 ASN 76 ASN 77 SER 78 VAL 81 LEU 82 GLY 83
ALA 74 ASN 76 ASN 77 SER 78 GLY 80 LEU 82 GLY 83
ALA 74 ASN 76 ASN 77 SER 78 GLY 80 VAL 81 GLY 83
ALA 74 ASN 76 ASN 77 SER 78 GLY 80 VAL 81 LEU 82
ALA 74 ASN 76 ASN 77 SER 78 ILE 79 LEU 82 GLY 83
ALA 74 ASN 76 ASN 77 SER 78 ILE 79 VAL 81 GLY 83
ALA 74 ASN 76 ASN 77 SER 78 ILE 79 VAL 81 LEU 82
ALA 74 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 GLY 83
ALA 74 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 LEU 82
ALA 74 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 VAL 81
ALA 74 LEU 75 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 74 LEU 75 SER 78 GLY 80 VAL 81 LEU 82 GLY 83
ALA 74 LEU 75 SER 78 ILE 79 VAL 81 LEU 82 GLY 83
ALA 74 LEU 75 SER 78 ILE 79 GLY 80 LEU 82 GLY 83
ALA 74 LEU 75 SER 78 ILE 79 GLY 80 VAL 81 GLY 83
ALA 74 LEU 75 SER 78 ILE 79 GLY 80 VAL 81 LEU 82
ALA 74 LEU 75 ASN 77 GLY 80 VAL 81 LEU 82 GLY 83
ALA 74 LEU 75 ASN 77 ILE 79 VAL 81 LEU 82 GLY 83
ALA 74 LEU 75 ASN 77 ILE 79 GLY 80 LEU 82 GLY 83


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ALA 74 LEU 75 ASN 77 ILE 79 GLY 80 VAL 81 GLY 83
ALA 74 LEU 75 ASN 77 ILE 79 GLY 80 VAL 81 LEU 82
ALA 74 LEU 75 ASN 77 SER 78 VAL 81 LEU 82 GLY 83
ALA 74 LEU 75 ASN 77 SER 78 GLY 80 LEU 82 GLY 83
ALA 74 LEU 75 ASN 77 SER 78 GLY 80 VAL 81 GLY 83
ALA 74 LEU 75 ASN 77 SER 78 GLY 80 VAL 81 LEU 82
ALA 74 LEU 75 ASN 77 SER 78 ILE 79 LEU 82 GLY 83
ALA 74 LEU 75 ASN 77 SER 78 ILE 79 VAL 81 GLY 83
ALA 74 LEU 75 ASN 77 SER 78 ILE 79 VAL 81 LEU 82
ALA 74 LEU 75 ASN 77 SER 78 ILE 79 GLY 80 GLY 83
ALA 74 LEU 75 ASN 77 SER 78 ILE 79 GLY 80 LEU 82
ALA 74 LEU 75 ASN 77 SER 78 ILE 79 GLY 80 VAL 81
ALA 74 LEU 75 ASN 76 GLY 80 VAL 81 LEU 82 GLY 83
ALA 74 LEU 75 ASN 76 ILE 79 VAL 81 LEU 82 GLY 83
ALA 74 LEU 75 ASN 76 ILE 79 GLY 80 LEU 82 GLY 83
ALA 74 LEU 75 ASN 76 ILE 79 GLY 80 VAL 81 GLY 83
ALA 74 LEU 75 ASN 76 ILE 79 GLY 80 VAL 81 LEU 82
ALA 74 LEU 75 ASN 76 SER 78 VAL 81 LEU 82 GLY 83
ALA 74 LEU 75 ASN 76 SER 78 GLY 80 LEU 82 GLY 83
ALA 74 LEU 75 ASN 76 SER 78 GLY 80 VAL 81 GLY 83
ALA 74 LEU 75 ASN 76 SER 78 GLY 80 VAL 81 LEU 82
ALA 74 LEU 75 ASN 76 SER 78 ILE 79 LEU 82 GLY 83
ALA 74 LEU 75 ASN 76 SER 78 ILE 79 VAL 81 GLY 83
ALA 74 LEU 75 ASN 76 SER 78 ILE 79 VAL 81 LEU 82
ALA 74 LEU ?5 ASN 76 SER 78 ILE 79 GLY 80 GLY 83
ALA 74 LEU 75 ASN 76 SER 78 ILE 79 GLY 80 LEU 82
ALA 74 LEU 75 ASN 76 SER 78 ILE 79 GLY 80 VAL 81
ALA 74 LEU 75 ASN 76 ASN 77 VAL 81 LEU 82 GLY 83
ALA 74 LEU 75 ASN 76 ASN 77 GLY 80 LEU 82 GLY 83
ALA 74 LEU 75 ASN ?6 ASN 77 GLY 80 VAL 81 GLY 83
ALA 74 LEU 75 ASN 76 ASN 77 GLY 80 VAL 81 LEU 82
ALA 74 LEU 75 ASN 76 ASN 77 ILE 79 LEU 82 GLY 83
ALA ?4 LEU 75 ASN 76 ASN 77 ILE 79 VAL 81 GLY 83
ALA 74 LEU 75 ASN 76 ASN 77 ILE 79 VAL 81 LEU 82
ALA 74 LEU 75 ASN 76 ASN 77 ILE 79 GLY 80 GLY 83
ALA 74 LEU 75 ASN 76 ASN 77 ILE 79 GLY 80 LEU 82


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ALA 74 LEU 75 ASN 76 ASN 77 ILE 79 GLY 80 VAL 81
ALA 74 LEU 75 ASN 76 ASN 77 SER 78 LEU 82 GLY 83
ALA 74 LEU 75 ASN 76 ASN 77 SER 78 VAL 81 GLY 83
ALA 74 LEU 75 ASN 76 ASN 77 SER 78 VAL 81 LEU 82
ALA 74 LEU 75 ASN 76 ASN 77 SER 78 GLY 80 GLY 83
ALA 74 LEU 75 ASN 76 ASN 77 SER 78 GLY 80 LEU 82
ALA 74 LEU 75 ASN 76 ASN 77 SER 78 GLY 80 VAL 81
ALA 74 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 GLY 83
ALA 74 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 LEU 82
ALA 74 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 VAL 81
ALA 74 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 GLY 80
ALA 73 SER 78 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ASN 77 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ASN 77 SER 78 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ASN 77 SER 78 ILE 79 VAL 81 LEU 82 GLY 83
ALA 73 ASN 77 SER 78 ILE 79 GLY 80 LEU 82 GLY 83
ALA 73 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 LEU 82
ALA 73 ASN 76 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ASN 76 SER 78 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ASN 76 SER 78 ILE 79 VAL 81 LEU 82 GLY 83
ALA 73 ASN 76 SER 78 ILE 79 GLY 80 LEU 82 GLY 83
ALA 73 ASN 76 SER 78 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 ASN 76 SER 781LE 79 GLY 80 VAL 81 LEU 82
ALA 73 ASN 76 ASN 77 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ASN 76 ASN 77 ILE 79 VAL 81 LEU 82 GLY 83
ALA 73 ASN 76 ASN 77 ILE 79 GLY 80 LEU 82 GLY 83
ALA 73 ASN 76 ASN 77 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 ASN 76 ASN 77 iLE 79 GLY 80 VAL 81 LEU 82
ALA 73 ASN 76 ASN 77 SER 78 VAL 81 LEU 82 GLY 83
ALA 73 ASN 76 ASN 77 SER 78 GLY 80 LEU 82 GLY 83
ALA 73 ASN 76 ASN 77 SER 78 GLY 80 VAL 81 GLY 83
ALA 73 ASN 76 ASN 77 SER 78 GLY 80 VAL 81 LEU 82
ALA 73 ASN 76 ASN 77 SER 78 ILE 79 LEU 82 GLY 83
ALA 73 ASN 76 ASN 77 SER 78 ILE 79 VAL 81 GLY 83
ALA 73 ASN 76 ASN 77 SER 78 ILE 79 VAL 81 LEU 82


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ALA 73 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 GLY 83
ALA 73 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 LEU 82
ALA 73 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 VAL 81
ALA 73 LEU 75 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 LEU ?5 SER 78 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 LEU 75 SER 78 ILE 79 VAL 81 LEU 82 GLY 83
ALA 73 LEU 75 SER 78 ILE 79 GLY 80 LEU 82 GLY 83
ALA 73 LEU 75 SER 78 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 LEU 75 SER 78 ILE 79 GLY 80 VAL 81 LEU 82
ALA 73 LEU 75 ASN 77 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 LEU 75 ASN 77 ILE 79 VAL 81 LEU 82 GLY 83
ALA 73 LEU 75 ASN 77 ILE 79 GLY 80 LEU 82 GLY 83
ALA 73 LEU 75 ASN 77 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 LEU 75 ASN 77 ILE 79 GLY 80 VAL 81 LEU 82
ALA 73 LEU 75 ASN 77 SER 78 VAL 81 LEU 82 GLY 83
ALA 73 LEU 75 ASN 77 SER 78 GLY 80 LEU 82 GLY 83
ALA 73 LEU 75 ASN 77 SER 78 GLY 80 VAL 81 GLY 83
ALA 73 LEU 75 ASN 77 SER 78 GLY 80 VAL 81 LEU 82
ALA 73 LEU 75 ASN 77 SER 78 ILE 79 LEU 82 GLY 83
ALA 73 LEU 75 ASN 77 SER 78 ILE 79 VAL 81 GLY 83
ALA 73 LEU 75 ASN 77 SER 78 ILE 79 VAL 81 LEU 82
ALA 73 LEU 75 ASN 77 SER 78 ILE 79 GLY 80 GLY 83
ALA 73 LEU 75 ASN 77 SER 78 ILE 79 GLY 80 LEU 82
ALA 73 LEU 75 ASN 77 SER 78 ILE 79 GLY 80 VAL 81
ALA 73 LEU 75 ASN 76 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 LEU 75 ASN 76 ILE 79 VAL 81 LEU 82 GLY 83
ALA 73 LEU 75 ASN 76 ILE 79 GLY 80 LEU 82 GLY 83
ALA 73 LEU 75 ASN 76 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 LEU 75 ASN 76 ILE 79 GLY 80 VAL 81 LEU 82
ALA 73 LEU 75 ASN 76 SER 78 VAL 81 LEU 82 GLY 83
ALA 73 LEU 75 ASN 76 SER 78 GLY 80 LEU 82 GLY 83
ALA ?3 LEU 75 ASN 76 SER 78 GLY 80 VAL 81 GLY 83
ALA 73 LEU 75 ASN 76 SER 78 GLY 80 VAL 81 LEU 82
ALA 73 LEU 75 ASN 76 SER 78 ILE 79 LEU 82 GLY 83
ALA 73 LEU 75 ASN 76 SER 78 ILE 79 VAL 81 GLY 83
ALA 73 LEU 75 ASN 76 SER 78 ILE 79 VAL 81 LEU 82


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ALA 73 LEU 75 ASN 76 SER 78 ILE 79 GLY 80 GLY 83
ALA 73 LEU 75 ASN 76 SER 78 ILE 79 GLY 80 LEU 82
ALA 73 LEU 75 ASN 76 SER 78 ILE 79 GLY 80 VAL 81
ALA 73 LEU 75 ASN 76 ASN 77 VAL 81 LEU 82 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 GLY 80 LEU 82 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 GLY 80 VAL 81 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 GLY 80 VAL 81 LEU 82
ALA 73 LEU 75 ASN 76 ASN 77 ILE 79 LEU 82 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 ILE 79 VAL 81 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 ILE 79 VAL 81 LEU 82
ALA 73 LEU 75 ASN 76 ASN 77 ILE 79 GLY 80 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 ILE 79 GLY 80 LEU 82
ALA 73 LEU 75 ASN 76 ASN 77 ILE 79 GLY 80 VAL 81
ALA 73 LEU 75 ASN 76 ASN 77 SER 78 LEU 82 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 SER 78 VAL 81 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 SER 78 VAL 81 LEU 82
ALA 73 LEU 75 ASN 76 ASN 77 SER 78 GLY 80 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 SER 78 GLY 80 LEU 82
ALA 73 LEU 75 ASN 76 ASN 77 SER 78 GLY 80 VAL 81
ALA 73 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 LEU 82
ALA 73 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 VAL 81
ALA 73 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 GLY 80
ALA 73 ALA 74 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 SER 78 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 SER 78 ILE 79 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 SER 78 ILE 79 GLY 80 LEU 82 GLY 83
ALA 73 ALA 74 SER 78 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 SER 78 ILE 79 GLY 80 VAL 81 LEU 82
ALA 73 ALA 74 ASN 77 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 ASN 77 ILE 79 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 ASN 771LE 79 GLY 80 LEU 82 GLY 83
ALA 73 ALA 74 ASN 77 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 ASN 77 ILE 79 GLY 80 VAL 81 LEU 82
ALA 73 ALA 74 ASN 77 SER 78 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 ASN 77 SER 78 GLY 80 LEU 82 GLY 83


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ALA 73 ALA 74 ASN 77 SER 78 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 ASN 77 SER 78 GLY 80 VAL 81 LEU 82
ALA 73 ALA 74 ASN 77 SER 78 ILE 79 LEU 82 GLY 83
ALA 73 ALA 74 ASN 77 SER 78 ILE 79 VAL 81 GLY 83
ALA 73 ALA 74 ASN 77 SER 78 ILE 79 VAL 81 LEU 82
ALA 73 ALA 74 ASN 77 SER 78 ILE 79 GLY 80 GLY 83
ALA 73 ALA 74 ASN 77 SER 78 ILE 79 GLY 80 LEU 82
ALA 73 ALA 74 ASN 77 SER 78 ILE 79 GLY 80 VAL 81
ALA 73 ALA 74 ASN 76 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 ASN 76 ILE 79 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 ASN 76 ILE 79 GLY 80 LEU 82 GLY 83
ALA 73 ALA 74 ASN 76 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 ASN 76 ILE 79 GLY 80 VAL 81 LEU 82
ALA 73 ALA 74 ASN 76 SER 78 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 ASN 76 SER 78 GLY 80 LEU 82 GLY 83
ALA 73 ALA 74 ASN 76 SER 78 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 ASN 76 SER 78 GLY 80 VAL 81 LEU 82
ALA 73 ALA 74 ASN 76 SER 78 ILE 79 LEU 82 GLY 83
ALA 73 ALA 74 ASN 76 SER 78 ILE 79 VAL 81 GLY 83
ALA 73 ALA 74 ASN 76 SER 78 ILE 79 VAL 81 LEU 82
ALA 73 ALA 74 ASN 76 SER 78 ILE 79 GLY 80 GLY 83
ALA 73 ALA 74 ASN 76 SER 78 ILE 79 GLY 80 LEU 82
ALA 73 ALA 74 ASN 76 SER 78 ILE 79 GLY 80 VAL 81
ALA 73 ALA 74 ASN 76 ASN 77 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 ASN 76 ASN 77 GLY 80 LEU 82 GLY 83
ALA 73 ALA 74 ASN 76 ASN 77 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 ASN 76 ASN 77 GLY 80 VAL 81 LEU 82
ALA 73 ALA 74 ASN 76 ASN 771LE 79 LEU 82 GLY 83
ALA 73 ALA 74 ASN 76 ASN 77 ILE 79 VAL 81 GLY 83
ALA 73 ALA 74 ASN 76 ASN 77 ILE 79 VAL 81 LEU 82
ALA 73 ALA 74 ASN 76 ASN 77 ILE 79 GLY 80 GLY 83
ALA 73 ALA 74 ASN 76 ASN 77 ILE 79 GLY 80 LEU 82
ALA 73 ALA 74 ASN 76 ASN 77 ILE 79 GLY 80 VAL 81
ALA 73 ALA 74 ASN 76 ASN 77 SER 78 LEU 82 GLY 83
ALA 73 ALA 74 ASN 76 ASN 77 SER 78 VAL 81 GLY 83
ALA 73 ALA 74 ASN 76 ASN 77 SER 78 VAL 81 LEU 82


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ALA 73 ALA 74 ASN 76 ASN 77 SER 78 GLY 80 GLY 83
ALA 73 ALA 74 ASN 76 ASN 77 SER 78 GLY 80 LEU 82
ALA 73 ALA 74 ASN 76 ASN 77 SER 78 GLY 80 VAL 81
ALA 73 ALA ?4 ASN 76 ASN 77 SER 781LE 79 GLY 83
ALA 73 ALA 74 ASN 76 ASN 77 SER 78 ILE 79 LEU $2
ALA 73 ALA 74 ASN 76 ASN 77 SER 78 ILE 79 VAL 81
ALA 73 ALA 74 ASN 76 ASN 77 SER 78 ILE 79 GLY 80
ALA 73 ALA 74 LEU 75 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ILE 79 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ILE 79 GLY 80 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 LEU 75 ILE 79 GLY 80 VAL 81 LEU 82
ALA 73 ALA 74 LEU 75 SER 78 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 SER 78 GLY 80 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 SER 78 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 LEU 75 SER 78 GLY 80 VAL 81 LEU 82
ALA 73 ALA 74 LEU 75 SER 78 ILE 79 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 SER 78 ILE 79 VAL 81 GLY 83
ALA 73 ALA 74 LEU 75 SER 78 ILE 79 VAL 81 LEU 82
ALA 73 ALA 74 LEU 75 SER 78 ILE 79 GLY 80 GLY 83
ALA 73 ALA 74 LEU 75 SER 78 ILE 79 GLY 80 LEU 82
ALA 73 ALA 74 LEU 75 SER 78 ILE 79 GLY 80 VAL 81
ALA 73 ALA 74 LEU 75 ASN 77 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 77 GLY 80 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 77 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 LEU 75 ASN 77 GLY 80 VAL 81 LEU 82
ALA 73 ALA 74 LEU 75 ASN 77 ILE 79 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 77 ILE 79 VAL 81 GLY 83
ALA 73 ALA 74 LEU 75 ASN 77 ILE 79 VAL 81 LEU 82
ALA 73 ALA 74 LEU 75 ASN 77 ILE 79 GLY 80 GLY 83
ALA 73 ALA 74 LEU 75 ASN 77 ILE 79 GLY 80 LEU 82
ALA 73 ALA 74 LEU 75 ASN 77 ILE 79 GLY 80 VAL 81
ALA 73 ALA 74 LEU 75 ASN 77 SER 78 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 77 SER 78 VAL 81 GLY 83
ALA 73 ALA 74 LEU 75 ASN 77 SER 78 VAL 81 LEU 82
ALA 73 ALA 74 LEU 75 ASN 77 SER 78 GLY 80 GLY 83


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ALA 73 ALA 74 LEU 75 ASN 77 SER 78 GLY 80 LEU 82
ALA 73 ALA 74 LEU 75 ASN 77 SER 78 GLY 80 VAL 81
ALA 73 ALA 74 LEU 75 ASN 77 SER 78 ILE 79 GLY 83
ALA 73 ALA 74 LEU 75 ASN 77 SER 78 ILE 79 LEU 82
ALA 73 ALA 74 LEU 75 ASN 77 SER 78 ILE 79 VAL 81
ALA 73 AI_.A 74 LEU 75 ASN 77 SER 78 ILE 79 GLY 80
ALA 73 ALA 74 LEU 75 ASN 76 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 GLY 80 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 GLY 80 VAL 81 LEU 82
ALA 73 ALA 74 LEU 75 ASN 76 ILE 79 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ILE 79 VAL 81 GLY 83
ALA 73 ALA 74 LEU 75 ASN 761LE 79 VAL 81 LEU 82
ALA 73 ALA 74 LEU 75 ASN 76 ILE 79 GLY 80 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ILE 79 GLY 80 LEU 82
ALA 73 ALA 74 LEU 75 ASN 76 ILE 79 GLY 80 VAL 81
ALA 73 ALA 74 LEU 75 ASN 76 SER 78 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 SER 78 VAL 81 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 SER 78 VAL 81 LEU 82
ALA 73 ALA 74 LEU 75 ASN 76 SER 78 GLY 80 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 SER 78 GLY 80 LEU 82
ALA 73 ALA 74 LEU 75 ASN 76 SER 78 GLY 80 VAL 81
ALA 73 ALA 74 LEU 75 ASN 76 SER 78 ILE 79 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 SER 78 ILE 79 LEU 82
ALA 73 ALA 74 LEU 75 ASN 76 SER 78 ILE 79 VAL 81
ALA 73 ALA 74 LEU 75 ASN 76 SER 78 ILE 79 GLY 80
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 VAL 81 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 VAL 81 LEU 82
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 GLY 80 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 GLY 80 LEU 82
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 GLY 80 VAL 81
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 ILE 79 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 ILE 79 LEU 82
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 ILE 79 VAL 81
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 ILE 79 GLY 80


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ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 SER 78 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 SER 78 LEU 82
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 SER 78 VAL 81
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 SER 78 GLY 80
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 SER 78 ILE 79
Table 9
Octuple Mutation Variants
ASN 76 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
LEU 75 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
LEU 75 ASN 76 SER 78 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
LEU 75 ASN 76 ASN 77 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
LEU 75 ASN 76 ASN 77 SER 78 GLY 80 VAL 81 LEU 82 GLY 83
LEU 75 ASN 76 ASN 77 SER 78 ILE 79 VAL 81 LEU 82 GLY 83
LEU 75 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 LEU 82 GLY 83
LEU 75 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 GLY 83
LEU 75 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 LEU 82
ALA 74 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 74 ASN 76 SER 78 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 74 ASN 76 ASN 77 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 74 ASN 76 ASN 77 SER 78 GLY 80 VAL 81 LEU 82 GLY 83
ALA 74 ASN 76 ASN 77 SER 78 ILE 79 VAL 81 LEU 82 GLY 83
ALA 74 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 LEU 82 GLY 83
ALA 74 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 GLY 83
ALA 74 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 LEU 82
ALA 74 LEU 75 SER 78 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 74 LEU 75 ASN 77 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 74 LEU 75 ASN 77 SER 78 GLY 80 VAL 81 LEU 82 GLY 83
ALA 74 LEU 75 ASN 77 SER 78 ILE 79 VAL 81 LEU 82 GLY 83
ALA 74 LEU 75 ASN 77 SER 78 ILE 79 GLY 80 LEU 82 GLY 83
ALA 74 LEU 75 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 GLY 83
ALA 74 LEU 75 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 LEU 82
ALA 74 LEU 75 ASN 76 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 74 LEU 75 ASN 76 SER 78 GLY 80 VAL 81 LEU 82 GLY 83
ALA 74 LEU 75 ASN 76 SER 78 ILE 79 VAL 81 LEU 82 GLY 83
ALA 74 LEU 75 ASN 76 SER 78 ILE 79 GLY 80 LEU 82 GLY 83


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ALA 74 LEU 75 ASN 76 SER 781LE 79 GLY 80 VAL 81 GLY 83
ALA 74 LEU 75 ASN 76 SER 78 ILE 79 GLY 80 VAL 81 LEU 82
ALA 74 LEU 75 ASN 76 ASN 77 GLY 80 VAL 81 LEU 82 GLY 83
ALA 74 LEU 75 ASN 76 ASN 77 ILE 79 VAL 81 LEU 82 GLY 83
ALA 74 LEU 75 ASN 76 ASN 77 ILE 79 GLY 80 LEU 82 GLY 83
ALA 74 LEU 75 ASN 76 ASN 77 ILE 79 GLY 80 VAL 81 GLY 83
ALA 74 LEU 75 ASN 76 ASN 77 ILE 79 GLY 80 VAL 81 LEU 82
ALA 74 LEU 75 ASN 76 ASN 77 SER 78 VAL 81 LEU 82 GLY 83
ALA 74 LEU 75 ASN 76 ASN 77 SER 78 GLY 80 LEU 82 GLY 83
ALA 74 LEU 75 ASN 76 ASN 77 SER 78 GLY 80 VAL 81 GLY 83
ALA 74 LEU 75 ASN 76 ASN 77 SER 78 GLY 80 VAL 81 LEU 82
ALA 74 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 LEU 82 GLY 83
ALA 74 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 VAL 81 GLY 83
ALA 74 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 VAL 81 LEU 82
ALA 74 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 GLY 83
ALA 74 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 LEU 82
ALA 74 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 VAL 81
ALA 73 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ASN 76 SER 78 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ASN 76 ASN 77 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ASN 76 ASN 77 SER 78 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ASN 76 ASN 77 SER 78 ILE 79 VAL 81 LEU 82 GLY 83
ALA 73 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 LEU 82 GLY 83
ALA 73 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 LEU 82
ALA 73 LEU 75 SER 78 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 LEU 75 ASN 77 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 LEU 75 ASN 77 SER 78 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 LEU 75 ASN 77 SER 78 ILE 79 VAL 81 LEU 82 GLY 83
ALA 73 LEU 75 ASN 77 SER 78 ILE 79 GLY 80 LEU 82 GLY 83
ALA 73 LEU 75 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 LEU 75 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 LEU 82
ALA 73 LEU 75 ASN 76 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 LEU 75 ASN 76 SER 78 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 LEU 75 ASN 76 SER 78 ILE 79 VAL 8I LEU 82 GLY 83
ALA 73 LEU 75 ASN 76 SER 78 ILE 79 GLY 80 LEU 82 GLY 83


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ALA 73 LEU 75 ASN 76 SER 78 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 LEU 75 ASN 76 SER 78 ILE 79 GLY 80 VAL 81 LEU 82
ALA 73 LEU 75 ASN 76 ASN 77 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 ILE 79 VAL 81 LEU 82 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 ILE 79 GLY 80 LEU 82 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 ILE 79 GLY 80 VAL 81 LEU 82
ALA 73 LEU 75 ASN 76 ASN 77 SER 78 VAL 81 LEU 82 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 SER 78 GLY 80 LEU 82 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 SER 78 GLY 80 VAL 81 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 SER 78 GLY 80 VAL 81 LEU 82
ALA 73 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 LEU 82 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 VAL 81 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 VAL 81 LEU 82
ALA 73 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 LEU 82
ALA 73 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 VAL 81
ALA 73 ALA 74 SER 78 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 ASN 77 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 ASN 77 SER 78 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 ASN 77 SER 78 ILE 79 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 ASN 77 SER 78 ILE 79 GLY 80 LEU 82 GLY 83
ALA 73 ALA 74 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 LEU 82
ALA 73 ALA 74 ASN 76 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 ASN 76 SER 78 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 ASN 76 SER 78 ILE 79 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 ASN 76 SER 78 ILE 79 GLY 80 LEU 82 GLY 83
ALA 73 ALA 74 ASN 76 SER 78 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 ASN 76 SER 78 ILE 79 GLY 80 VAL 81 LEU 82
ALA 73 ALA 74 ASN 76 ASN 77 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 ASN 76 ASN 77 ILE 79 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 ASN 76 ASN 77 ILE 79 GLY 80 LEU 82 GLY 83
ALA 73 ALA 74 ASN 76 ASN 77 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 ASN 76 ASN 77 ILE 79 GLY 80 VAL 8 i LEU 82
ALA 73 ALA 74 ASN 76 ASN 77 SER 78 VAL 81 LEU 82 GLY 83


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ALA 73 ALA 74 ASN 76 ASN 77 SER 78 GLY 80 LEU 82 GLY 83
ALA 73 ALA 74 ASN ?6 ASN 77 SER 78 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 ASN 76 ASN 77 SER 78 GLY 80 VAL 81 LEU 82
ALA 73 ALA 74 ASN 76 ASN 77 SER 78 ILE 79 LEU 82 GLY 83
ALA 73 ALA 74 ASN 76 ASN 77 SER 78 ILE 79 VAL 81 GLY 83
ALA 73 ALA 74 ASN 76 ASN 77 SER 78 ILE 79 VAL 81 LEU 82
ALA 73 ALA 74 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 GLY 83
ALA 73 ALA 74 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 LEU 82
ALA 73 ALA 74 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 VAL 81
ALA 73 ALA 74 LEU 75 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 SER 78 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 SER 781LE 79 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 SER 78 ILE 79 GLY 80 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 SER 78 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 LEU 75 SER 78 ILE 79 GLY 80 VAL 81 LEU 82
ALA 73 ALA 74 LEU 75 ASN 77 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 77 ILE 79 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 77 ILE 79 GLY 80 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 77 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 LEU 75 ASN 77 ILE 79 GLY 80 VAL 81 LEU 82
ALA 73 ALA 74 LEU 75 ASN 77 SER 78 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 77 SER 78 GLY 80 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 77 SER 78 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 LEU 75 ASN 77 SER 78 GLY 80 VAL 81 LEU 82
ALA 73 ALA 74 LEU 75 ASN 77 SER 78 ILE 79 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 77 SER 78 ILE 79 VAL 81 GLY 83
ALA 73 ALA 74 LEU 75 ASN 77 SER 78 ILE 79 VAL 81 LEU 82
ALA 73 ALA 74 LEU 75 ASN 77 SER 78 ILE 79 GLY 80 GLY 83
ALA 73 ALA 74 LEU 75 ASN 77 SER 78 ILE 79 GLY 80 LEU 82
ALA 73 ALA 74 LEU 75 ASN 77 SER 78 ILE 79 GLY 80 VAL 81
ALA 73 ALA 74 LEU 75 ASN 76 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ILE 79 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ILE 79 GLY 80 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ILE 79 GLY 80 VAL 81 LEU 82
ALA 73 ALA 74 LEU 75 ASN 76 SER 78 VAL 81 LEU 82 GLY 83


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ALA 73 ALA 74 LEU 75 ASN 76 SER 78 GLY 80 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 SER ?8 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 SER 78 GLY 80 VAL 81 LEU 82
ALA 73 ALA 74 LEU 75 ASN 76 SER 78 ILE 79 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 SER 78 ILE 79 VAL 81 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 SER 78 ILE 79 VAL 81 LEU 82
ALA 73 ALA 74 LEU 75 ASN ?6 SER 78 ILE 79 GLY 80 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 SER 78 ILE 79 GLY 80 LEU 82
ALA 73 ALA 74 LEU 75 ASN 76 SER 78 ILE 79 GLY 80 VAL 81
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 GLY 80 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 GLY 80 VAL 81 LEU 82
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 ILE 79 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 ILE 79 VAL 81 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 ILE 79 VAL 81 LEU 82
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 ILE 79 GLY 80 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 ILE 79 GLY 80 LEU 82
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 ILE 79 GLY 80 VAL 81
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 SER 78 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 SER 78 VAL 81 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 SER 78 VAL 81 LEU 82
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 SER 78 GLY 80 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 SER 78 GLY 80 LEU 82
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 SER 78 GLY 80 VAL 81
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 LEU 82
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 VAL 81
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 GLY 80
Table 10
Nonuple Mutation Variants
LEU 75 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 74 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 74 LEU 75 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 74 LEU 75 ASN 76 SER 78 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83


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ALA 74 LEU 75 ASN 76 ASN 77 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 74 LEU 75 ASN 76 ASN 77 SER 78 GLY 80 VAL 81 LEU 82 GLY 83
ALA 74 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 VAL 81 LEU 82 GLY 83
ALA 74 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 LEU 82 GLY 83
ALA 74 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 GLY 83
ALA 74 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 LEU 82
ALA 73 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 LEU 75 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 LEU 75 ASN 76 SER 78 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 SER 78 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 VAL 81 LEU 82 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 LEU 82 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 LEU 82
ALA 73 ALA 74 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 ASN 76 SER 78 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 ASN 76 ASN 77 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 ASN 76 ASN 77 SER 78 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 ASN 76 ASN 77 SER 78 ILE 79 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 LEU 82 GLY 83
ALA 73 ALA 74 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 LEU 82
ALA 73 ALA 74 LEU 75 SER 78 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 77 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 77 SER 78 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 77 SER 78 ILE 79 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 77 SER 78 ILE 79 GLY 80 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 LEU 75 ASN 77 SER 78 ILE 79 GLY 80 VAL 81 LEU 82
ALA 73 ALA 74 LEU 75 ASN 76 ILE 79 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 SER 78 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 SER 78 ILE 79 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 SER 78 ILE 79 GLY 80 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 SER 78 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 SER 78 ILE 79 GLY 80 VAL 81 LEU 82


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64
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 GLY 80 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 ILE 79 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 ILE 79 GLY 80 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 ILE 79 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 ILE 79 GLY 80 VAL 81 LEU 82
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 SER 78 VAL 81 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 SER 78 GLY 80 LEU 82 GLY 83
ALA 73 ALA '74 LEU 75 ASN 76 ASN 77 SER 78 GLY 80 VAL 81 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 SER 78 GLY 80 VAL 81 LEU 82
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 LEU 82 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 SER 78 ILE ?9 VAL 81 GLY 83
ALA ?3 ALA 74 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 VAL 81 LEU 82
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 GLY 83
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 LEU 82
ALA 73 ALA 74 LEU 75 ASN 76 ASN 77 SER 78 ILE 79 GLY 80 VAL 81
Table 11 exemplifies non-limiting preferred double mutation variants having
one
or more deletions and one or more substitutions:
Table 11
0 73, Leu75lle D 73, Leu75Met


O 73, Asn76His D 73, Ser78Asn


D 73, Ser78Thr O 73, Ile79Leu


D 73, Ile79Met 0 73, Ile79Thr


D 73, Va181Thr D 73, Leu82Phe


O 73, Leu82His 0 74, Leu82His


D 74, Leu82Phe D 74, Va181Thr


O 74, Va181 Thr 0 74, Ile79Thr


O 74, Ile79Met O 74, Ile79Leu


O 74, Ser78Thr D 74, Ser78Asn


0 74, Asn76His O 74, Leu75Met


0 74, Leu75Ile 0 74, Ser78Asp


O 75, Asn76His D 75, Ser78Asn




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D 75, Ser78Thr D 75, Ile79Leu


O 75, Ile79Met O 75, Ile79Thr


0 75, Va181Thr O 75, Leu82Phe


0 75, Leu82His D 76, Leu75Ile


D 76, Leu75Met 0 76, Ser78Asn


D 76, Leu75Thr 0 76, Ile79Leu


0 76, Ile79Met D 76, Ile79Thr


O 76, Va181Thr O 76, Leu82Phe


D 76, Leu82His ~ 77, Leu75Ile


O 77, Leu75Met O 77, Asn76His


D 77, Ser78Asn D 77, Ser78Thr


0 77, Ile79Leu D 77, Ile79Met


D 77, Ile79Thr O 77, Va181Thr


O 77, Leu82Phe O 77, Leu82His


D 78, Leu82His D 78, Leu75Ile


O 78, Leu75Met D 78, Leu75Ile


0 78, Asn76His O 78, Ile79Leu


D 78, Ile79Met D 78, Ile79Thr


0 78, Va181Thr ~ 78, Leu82Phe


0 79, Leu82His D 79, Leu75Ile


O 79, Leu75Met O 79, Asn76His


D 79, Ser78Asn D 79, Ser78Thr


O 79, Va181Thr O 79, Leu82Phe


O 80, Leu75Ile O 80, Leu75Met


D 80, Asn76His 0 80, Ser78Asn


D 80, Ser78Thr O 80, Ile79Leu


D 80, Ile79Met 0 80, Ile79Thr


D 80, Va181Thr O 80, Leu82Phe


D 80, Leu75Ile 0 81, Leu75Ile




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66
O 81, Leu7~Met D 81, Asn76His


D 81, Ser78Asn 0 81, Ser78Thr


D 81, IIe79Leu D 81, Ile79Met


0 81, IIe79Thr 0 81, Leu82Phe


O 81, Leu82His O 82, Leu75Ile


O 82, Leu75Met D 82, Asn76His


O 82, Ser78Asn O 82, Ser78Thr


D 82, IIe79Leu O 82, IIe79Met


O 82, Ile79Thr D 82, Va181Thr


O 83, Leu75Ile D 83, Leu75Met


O 83, Asn76His D 83, Ser78Asn


O 83, Ser78Thr O 83, Ile79Leu


O 83, IIe79Met ~ 83, IIe79Thr


D 83, Va181Thr O 83, Leu82Phe


One or more additional substitution mutations ("stabilizing substitutions")
outside the epitope region may additionally be made in order to, for example,
restabilize
the protease upon mutation of the epitope region or to enhance the proteolytic
activity of
the variant. Many such stabilizing mutations are well known in the art.
Examples of
such stabilizing mutations are disclosed in, for example, WO 95/10591, Baeck
et al.,
published April 20, 1995; U.S. Pat. No. 4,914,031, Zukowski et al., issued
April 3, 1990;
U.S. Pat. No. 5,470,733, Bryan et al., issued November 28, 1995; U.S. Pat. No.
5,567,601, Bran et al., issued October 22, 1996; WO 89/07642, U.S. Pat. No.
5,707,848, Bryan et al., issued January 13, 1998; Van Eekelen et al..
published August
24, 1989; WO 87/04461, Stabinsky et al.. published July 30, 1987; U.S. Pat.
No.
4,760,025, Estell et al., issued July 26, 1988; WO 92/11348, Branner et al..
published
July 9, 1992; EP 0,405,901, Casteleiin et al.. published January 2, 1991; WO
91/00345,
Branner et al., published January 10, 1991; and WO 94/10020, Brode et al..
published
March 23, 1995.


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67
Preferred stabilizing mutations include one or more of I107V; K213R; Y217L;
Y217K; N218S; G169A; MSOF; Q19E; PSA; S9A; I31L; E156S; G169A; N212G;
S188P; T254A; S3C + Q206C; and Q271E. The more preferred stabilizing mutations
include one or more of PSA; S9A; I31L; E156S; G169A; N212G; S188P; T254A; S3C
+
Q206C; Q271 E; Y217L; and Y217K. The most preferred stabilizing mutations
include
Y217L and Y217K.
A preferred variant for use in personal care compositions is O 75 - 83, Y217L.
Method of Makins
The variants are prepared by mutating the nucleotide sequences that code for a
wild-type serine protease, thereby resulting in variants having modified amino
acid
sequences. Such methods are well-known in the art; one such method is set
forth below:
A phagemid (pSS-5) containing the wild-type subtilisin BPN' gene (Mitchison,
C. and J.A. Wells, "Protein Engineering of Disulfide Bonds in Subtilisin
BPN"',
Biochemistry, Vol. 28, pp. 4807 - 4815 (1989) is transformed into Escherichia
coli dut-
ung- strain CJ236 and a single stranded uracil-containing DNA template is
produced
using the VCSM13 helper phage (Kunkel et al., "Rapid and Efficient Site-
Specific
Mutagenesis Without Phenotypic Selection", Methods in Enzymology, Vol 154, pp.
367
- 382 (1987), as modified by Yuckenberg et al., "Site-Directed in vitro
Mutagenesis
Using Uracil-Containing DNA and Phagemid Vectors", Directed Mutagenesis - A
Practical Ap rp oach. McPherson, M. J. ed., pp. 27 - 48 (1991). Primer site-
directed
mutagenesis modified from the method of Zoller and Smith (Zoller, M. J., and
M. Smith,
"Oligonucleotide - Directed Mutagenesis Using M13 - Derived Vectors: An
Efficient
and General Procedure for the Production of Point Mutations in any Fragment of
DNA",
Nucleic Acids Research, Vol. 10, pp. 6487 - 6500 (1982) is used to produce all
mutants
(essentially as presented by Yuckenberg et al., supra}.
Oligonucleotides are made using a 380B DNA synthesizer (Applied Biosystems
Inc.). Mutagenesis reaction products are transformed into Escherichia coli
strain
MM294 (American Type Culture Collection E. coli 33625). All mutations are.
confirmed by DNA sequencing and the isolated DNA is transformed into the
Bacillus
subtilis expression strain PG632 (Saunders et al., "Optimization of the Signal-
Sequence


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68
Cleavage Site for Secretion from Bacillus subtilis of a 34-amino acid Fragment
of
Human Parathyroid Hormone", Gene, Vol. 102, pp. 277 - 282 (1991) and Yang et
al.,
"Cloning of the Neutral Protease Gene of Bacillus subtilis and the Use of the
Cloned
Gene to Create an in vitro - Derived Deletion Mutation", Journal of
Bacteriology, Vol.
160, pp. 1 S - 21 (1984). Preliminary assessment of variant activity is
determined by the
ability of PG632 cells transformed with mutant plasmids to hydrolyze casein.
Fermentation is as follows. Bacillus subtilis cells (PG632) containing the
variant
of interest are grown to mid-log phase in one liter of LB broth containing 10
g/L
glucose, and inoculated into a Biostat C fermentor (Braun Biotech, Inc.,
Allentown, PA)
in a total volume of 9 liters. The fermentation medium contains yeast extract,
casein
hydrosylate, soluble - partially hydrolyzed starch (Maltrin M-250), antifoam,
buffers,
and trace minerals see "Biology of Bacilli: Applications to Industry", Doi, R.
H. and M.
McGloughlin, eds. (1992)). The broth is kept at a constant pH of 7.5 during
the
fermentation run. Kanamycin (50 pg/mL) is added for antibiotic selection of
the
mutagenized plasmid. The cells are grown for 18 hours at 37 °C to an A~
of about 60
and the product harvested.
The fermentation broth is taken through the following steps to obtain pure
variant. The broth is cleared of Bacillus subtilis cells by tangential flow
against a 0.16
p,m membrane. The cell-free broth is then concentrated by ultrafiltration with
a 8000
molecular weight cut-off membrane. The pH is adjusted to 5.5 with concentrated
MES
buffer (2-(N-morpholino~thanesulfonic acid). The variant is further purified
by cation
exchange chromatography with S-sepharose and elution with NaCI gradients. see
Scopes, R. K., "Protein Purification Principles and Practice", Springer-
Verlag, New
York (1984).
A pNA assay (DelMar et al., Analytical Biochemistry, Vol. 99, pp. 316 - 320
(1979)) is used to determine the active variant concentration for fractions
collected
during gradient elution. This assay measures the rate at which p-nitroaniline
is released
as the variant hydrolyzes the soluble synthetic substrate, succinyl-alanine-
alanine-
proline-phenylalanine p-nitroaniline (sAAPF pNA). The rate of production of
yellow .
color from the hydrolysis reaction is measured at 410 nm on a
spectrophotometer and is


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69
proportional to the active enzyme concentration. In addition, absorbance
measurements
at 280 nm are used to determine the total protein concentration. The active
enzyme/total-protein ratio gives the variant purity, and is used to identify
fractions to be
pooled for the stock solution.
To avoid autolysis of the variant during storage, an equal weight of propylene
glycol is added to the pooled fractions obtained from the chromatography
column. Upon
completion of the purification procedure the purity of the stock variant
solution is
checked with SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel
electrophoresis)
and the absolute enzyme concentration is determined via an active site
titration method
using trypsin inhibitor type II-T: turkey egg white (Sigma Chemical Company,
St.
Louis, MO).
In preparation for use, the enzyme stock solution is eluted through a Sephadex-

625 (Pharmacia, Piscataway, New Jersey) size exclusion column to remove the
propylene glycol and exchange the buffer. The MES buffer in the enzyme stock
solution
is exchanged for 0.1 M tris buffer (tris(hydroxymethyl-aminomethane)
containing
O.O1M CaClz and pH adjusted to 8.6 with HCI. All experiments are carned out at
pH 8.6
in tris buffer thermostated at 25 °C.
Analytical Methods
The present variants may be tested for enzymatic activity and immune and / or
allergenic response using the following methods, both of which are known to
one skilled
in the art. Alternatively, other methods well-known in the art may be used.
Variant Activity
The protease activity of a variant of the present invention may be assayed by
methods which are well-known in the art. Two such methods are set forth herein
below:
Skin Flake Activity Method
Using Scotch~ #37506 tape, human skin flakes are stripped from the legs of a
subject repeatedly until the tape is substantially opaque with flakes. The
tape is then cut.
into 1 inch by 1 inch squares and set aside. In a 10 mm by 35 mm petri dish, 2
mL of
0.75 mg / mL of a control enzyme (for example, subtilisin BPN') or the variant
to be


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tested is added in 0.01 M KH2P04 pH 5.5 buffer. To this solution 1 mL of 2.5%
sodium
laurate pH 8.6 solution is added. The solution is gently mixed on a platform
shaker.
The previously prepared tape square is soaked in the solution (flake side up)
for ten
minutes continuing gentle mixing. The tape square is then rinsed gently in tap
water for
fifteen seconds. Stevenel Blue Stain (3 mL, commercially available from Sigma
Chemical Co., St. Louis, MO) is pipetted into a clean petri dish. 'The rinsed
tape square
is placed into the stain for three minutes (flake side up) with gentle mixing.
The tape
square is removed from the stain and rinsed consecutively in two beakers of
300 mL
distilled water, for fifteen seconds per rinse. The tape square is allowed to
air-dry. The
color intensity between the tape square obtained from the control enzyme and
the tape
square obtained from the variant is compared visually or by using a
chromameter.
Relative to the control enzyme tape square, a variant tape square showing less
color
intensity is indicative of a variant having higher activity.
Dved Collagen Activit~Method
Combine 50 mL of 0.1 M tris buffer (tris-hydroxymethyl-aminomethane)
containing 0.01 M CaCl2 to give pH 8.6, and 0.5 g azocoll (azo dye impregnated
collagen, commercially available from Sigma Chemical Co., St. Louis, MO).
Incubate
this mixture at 25 °C while gently mixing with a platform shaker.
Filter 2 mL of the
mixture through a 0.2 micron syringe filter and read absorbance of the mixture
at 520
nm to zero a spectrophotometer. Add 1 ppm of a control enzyme (for example,
subtilisin
BPN') or the variant to be tested to the remaining 48 mL of tris / azocoll
mixture. Filter
2 mL of the control / variant containing solution through a 0.2 micron syringe
filter every
two minutes for a total of ten minutes. For each filtered sample, read the
absorbance
immediately at 520 nm. Plot the results against time. The slopes of the
control and the
test conjugate are indicative of relative activities of the samples. A higher
slope is
indicative of a higher activity. The test variant activity (slope) may be
expressed as a
percent of the control activity (slope).
T-Cell Proliferation Assay
The immune and / or allergenic potential of the variants of the present
invention
may be determined using a T-cell proliferation assay such as the assay
presented


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71
hereinbelow. This assay is a variation of the assay disclosed in Bunay Poor
Fard et al.,
"T Cell Epitopes of the Major Fraction of Rye Grass Lolium perenne (Lol p I)
Defined
Using Overlapping Peptides in vitro and in vivo", Clinical Experimental
Immunology,
Vol. 94, pp. 111 - 116 (1993).
The blood of subjects allergic to subtilisin BPN' (prick test positive) and
control
subjects (prick test negative) are used in this assay. Blood (~ 60 mL) from
each subject
is collected and mononuclear cells are harvested using ficoll-hypaque (which
may be
obtained from Pharmacia, Piscataway, New Jersey). The cells are washed twice
in
RPMI 1640 (which may be obtained from Gibco, Grand Island, New York) and then
resuspended in complete medium RPMI supplemented with 10% human AB-serum, 2
mM L-glutamine, and 25 pg / mL gentamicin (which may be obtained from Gibco).
Cells are cultured at a concentration of 2 x 105 cells / well in 0.2 mL of
complete
medium in U-bottomed 96-well microtiter plates. The potential antigen to be
tested
(either inactivated BPN' as positive control or a variant of the present
invention) is
added at a final concentration up to about 40 pg / mL. Cultures are incubated
at 37 °C in
5% C02. After five days, 1 p,Ci / well of methyl-3H-thymidine is added and 18
hours
later the cells are harvested. 3H-thymidine incorporation by the cell is
assessed as a
measure of T-cell proliferation by liquid scintillation counting.
Compositions of the Present Invention
The variants may be utilized in cleaning compositions including, but not
limited
to, laundry compositions, hard surface cleansing compositions, light duty
cleaning
compositions including dish cleansing compositions, and automatic dishwasher
detergent compositions.
The cleaning compositions herein comprise an effective amount of one or more
variants of the present invention and a cleaning composition Garner.
As used herein, "effective amount of variant", or the like, refers to the
quantity of
variant necessary to achieve the proteolytic activity necessary in the
specific cleaning
composition. Such effective amounts are readily ascertained by one of ordinary
skill in
the art and is based on many factors, such as the particular variant used, the
cleaning
application; the specific composition of the cleaning composition, and whether
a liquid


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72
or dry (e.g., granular, bar) composition is desired, and the like. Preferably,
the cleaning
compositions comprise from about 0.0001 % to about 10%, more preferably from
about
0.001 % to about 1 %, and most preferably from about 0.01 % to about 0.1 % of
one or
more variants of the present invention. Several examples of various cleaning
compositions wherein the variants may be employed are discussed in further
detail
below.
In addition to the present variants, the present cleaning compositions further
comprise a cleaning composition carrier comprising one or more cleaning
composition
materials compatible with the variant. The term "cleaning composition
material", as
used herein, means any material selected for the particular type of cleaning
composition
desired and the form of the product (e.g., liquid, granule, bar, spray, stick,
paste, gel),
which materials are also compatible with the variant used in the composition.
The
specific selection of cleaning composition materials is readily made by
considering the
material to be cleaned, the desired form of the composition for the cleaning
condition
during use. The term "compatible", as used herein, means the cleaning
composition
materials do not reduce the proteolytic activity of the variant to such an
extent that the
variant is not effective as desired during normal use situations. Specific
cleaning
composition materials are exemplified in detail hereinafter.
The variants of the present invention may be used in a variety of detergent
compositions where high sudsing and good cleansing activity is desired. Thus,
the
variants can be used with various conventional ingredients to provide fully-
formulated
hard-surface cleaners, dishwashing compositions, fabric laundering
compositions, and
the like. Such compositions can be in the form of liquids, granules, bars, and
the like.
Such compositions can be formulated as "concentrated" detergents which contain
as
much as from about 30% to about 60% by weight of surfactants.
The cleaning compositions herein may optionally, and preferably, contain
various surfactants (e.g., anionic, nonionic, or zwitterionic surfactants).
Such surfactants
are typically present at levels of from about 5% to about 35% of the
compositions.
Nonlimiting examples of surfactants useful herein include the conventional C"-
C 1 g alkyl benzene sulfonates and primary and random alkyl sulfates, the C 1
p-C 18


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73
secondary (2,3) alkyl sulfates of the formulas CH3(CH2)x(CHOS03)'M+)CH3 and
CH3(CH2)y(CHOS03-M+) CH2CH3 wherein x and (y+1) are integers of at least about
7, preferably at least about 9, and M is a water-solubilizing cation,
especially sodium,
the C 10-C 1 g alkyl alkoxy sulfates (especially EO 1-5 ethoxy sulfates), C 1
p-C 1 g alkyl
alkoxy carboxylates (especially the EO 1-5 ethoxycarboxylates), the C 10-C 1 g
alkyl
polyglycosides, and their corresponding sulfated polyglycosides, C 12-C 1 g a-
sulfonated
fatty acid esters, C 12-C 1 g alkyl and alkyl phenol alkoxylates (especially
ethoxylates and
mixed ethoxy/propoxy), C 12-C 1 g betaines and sulfobetaines ("sultaines"), C
10-C 18
amine oxides, and the like. The alkyl alkoxy sulfates (AES) and alkyl alkoxy
carboxylates (AEC) are preferred herein. The use of such surfactants in
combination
with the amine oxide and / or betaine or sultaine surfactants is also
preferred, depending
on the desires of the formulator. Other conventional useful surfactants are
listed in
standard texts. Particularly useful surfactants include the C 10-C 1 g N-
methyl. glucamides
disclosed in U.S. Pat. No. 5, 194,639, Connor et al., issued March 16, 1993.
A wide variety of other ingredients useful in detergent cleaning compositions
can
be included in the compositions herein including, for example, other active
ingredients,
carriers, hydrotropes, processing aids, dyes or pigments, and solvents for
liquid
formulations. If an additional increment of sudsing is desired, suds boosters
such as the
C 10-C 16 alkolamides can be incorporated into the compositions, typically at
about 1 % to
about 10% levels. The C 10-C 14 monoethanol and diethanol amides illustrate a
typical
class of such suds boosters. Use of such suds boosters with high sudsing
adjunct
surfactants such as the amine oxides, betaines and sultaines noted above is
also
advantageous. If desired, soluble magnesium salts such as MgCl2, MgS04, and
the like,
can be added at levels of, typically, from about 0.1% to about 2%, to provide
additional
sudsing.
The liquid detergent compositions herein may contain water and other solvents
as carriers. Low molecular weight primary or secondary alcohols exemplified by
methanol, ethanol, propanol, and iso-propanol are suitable. Monohydric
alcohols are
preferred for solubilizing surfactants, but polyols such as those containing
from about 2
to about 6 carbon atoms and from about 2 to about 6 hydroxy groups (e.g., 1,3-


CA 02324422 2000-09-22
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74
propanediol, ethylene glycol, glycerine, and 1,2-propanediol) can also be
used. The
compositions may contain from about S% to about 90%, typically from about 10%
to
about 50% of such carriers.
The detergent compositions herein will preferably be formulated such that
during
use in aqueous cleaning operations, the wash water will have a pH between
about 6.8
and about 11. Finished products are typically formulated at this range.
Techniques for
controlling pH at recommended usage levels include the use of, for example,
buffers,
alkalis, and acids. Such techniques are well known to those skilled in the
art.
When formulating the hard surface cleaning compositions and fabric cleaning
compositions of the present invention, the formulator may wish to employ
various
builders at levels from about S% to about 50% by weight. Typical builders
include the
1-10 micron zeolites, polycarboxylates such as citrate and oxydisuccinates,
layered
silicates, phosphates, and the like. Other conventional builders are listed in
standard
formularies.
Likewise, the formulator may wish to employ various additional enzymes, such
as cellulases, lipases, amylases and proteases in such compositions, typically
at levels of
from about 0.001% to about 1% by weight. Various detersive and fabric care
enzymes
are well-known in the laundry detergent art.
Various bleaching compounds, such as the percarbonates, perborates and the
like,
can be used in such compositions, typically at levels from about 1% to about
15% by
weight. If desired, such compositions can also contain bleach activators such
as
tetraacetyl ethylenediamine, nonanoyloxybenzene sulfonate, and the like, which
are also
known in the art. Usage levels typically range from about 1 % to about 10% by
weight.
Soil release agents, especially of the anionic oligoester type, chelating
agents,
especially the aminophosphonates and ethylenediaminedisuccinates, clay soil
removal
agents, especially ethoxylated tetraethylene pentamine, dispersing agents,
especially
polyacrylates and polyasparatates, brighteners, especially anionic
brighteners, suds
suppressors, especially silicones and secondary alcohols, fabric softeners,
especially
smectite clays, and the like can all be used in such compositions at levels
ranging from


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about 1 % to about 35% by weight. Standard formularies and published patents
contain
multiple, detailed descriptions of such conventional materials.
Enzyme stabilizers may also be used in the cleaning compositions. Such enzyme
stabilizers include propylene glycol (preferably from about 1 % to about 10%),
sodium
formate (preferably from about 0. I % to about 1 %) and calcium formate
(preferably from
about 0.1 % to about 1 %).
The present variants are useful in hard surface cleaning compositions. As used
herein "hard surface cleaning composition" refers to liquid and granular
detergent
compositions for cleaning hard surfaces such as floors, walls, bathroom tile,
and the like.
Hard surface cleaning compositions of the present invention comprise an
effective
amount of one or more variants of the present invention, preferably from about
0.001
to about 10%, more preferably from about 0.01 % to about 5%, more preferably
still from
about 0.05% to about 1 % by weight of variant of the composition. In addition
to
comprising one or more of the variants, such hard surface cleaning
compositions
typically comprise a surfactant and a water-soluble sequestering builder. In
certain
specialized products such as spray window cleaners, however, the surfactants
are
sometimes not used since they may produce a filmy and / or streaky residue on
the glass
surface.
The surfactant component, when present, may comprise as little as 0.1 % of the
compositions herein, but typically the compositions will contain from about
0.25% to
about 10%, more preferably from about 1% to about 5% of surfactant.
Typically the compositions will contain from about 0.5% to about 50% of a
detergency builder, preferably from about 1 % to about 10%.
Preferably the pH should be in the range of from about 7 to about 12.
Conventional pH adjustment agents such as sodium hydroxide, sodium carbonate
or
hydrochloric acid can be used if adjustment is necessary.
Solvents may be included in the compositions. Useful solvents include, but are
not limited to, glycol ethers such as diethyleneglycol monohexyl ether,
diethyleneglycol
monobutyl ether, ethyleneglycol monobutyl ether, ethyleneglycol monohexyl
ether,
propyleneglycol monobutyl ether, dipropyleneglycol monobutyl ether, and diols
such as


CA 02324422 2000-09-22
WO 99/49057 PCT/IB99/00520
76
2,2,4-trimethyl-1,3-pentanediol and 2-ethyl-1,3-hexanediol. When used, such
solvents
are typically present at levels of from about 0.5% to about 15%, more
preferably from
about 3% to about 11%.
Additionally, highly volatile solvents such as iso-propanol or ethanol can be
used
in the present compositions to facilitate faster evaporation of the
composition from
surfaces when the surface is not rinsed after "full strength" application of
the
composition to the surface. When used, volatile solvents are typically present
at levels
of from about 2% to about 12% in the compositions.
Hard surface cleaning compositions of the present invention are illustrated by
the
following examples.
Examples 1 - 6
Liquid Hard Surface Cleaning Compositions
Ex.l Ex.2 Ex.3 Ex.4 Ex.S Ex.6


O 75 - 82 0.05 0.50 0.02 0.03 0.30 0.05
% % % % %


EDTA - - 2.90 2.90 - -
% %


Sodium Citrate - - - - 2.90 2.90
%


NaC,2 Alkyl-benzene1.95 - 1.95 - 1.95 -
sulfonate % % %


NaC,2 Alkylsulfate- 2.20 - 2.20 - 2.20
% %


NaC,Z (ethoxy) - 2.20 - 2.20 - 2.20
sulfate % %


C,Z Dimethylamine- 0.50 - 0.50 - 0.50
oxide % %


Sodium cumene 1.30 - 1.30 - 1.30 -
sulfonate % % %


Hexyl Carbitol 6.30 6.30 6.30 6.30 6.30 6.30
% % % % %


Water 90.4 88.3 87.53 85.87 87.25 85.85
% %


All formulas are adjusted to pH 7.


CA 02324422 2000-09-22
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77
In Examples 1 - 6, the variants recited in Tables 2 - 10, and the preferred
variants
cited herein, among others, are substituted for O 75 - 82, with substantially
similar
results.
In another embodiment of the present invention, dishwashing compositions
comprise one or more variants of the present invention. As used herein,
"dishwashing
composition" refers to all forms of compositions for cleaning dishes
including, but not
limited to, granular and liquid forms. Dishwashing compositions of the present
invention are illustrated by the following examples.
Examples 7 - 10
Liquid Dish Detergent
Ex.7 Ex.8 Ex.9 Ex.lO


D79 0.05% 0.50% 0.02% 0.40%


C,Z- C" N-methyl glucamide0.90 0.90 % 0.90 0.90
% %


C, Z ethoxy ( 1 ) sulfate12.0 I 2.0 12.0 12.0
% % %


2-Methyl undecanoic acid 4.50 4.50 % 4.50 4.50
% %


C,Z ethoxy (2) carboxylate4.50 4.50 % 4.50 4.50
% %


C,z alcohol ethoxylate 3.00 3.00 % 3.00 3.00
(4) % %


C,2 amine oxide 3.00 3.00 % 3.00 3.00
% %


Sodium cumene sulfonate 2.00 2.00 % 2.00 2.00
% %


Ethanol 4.00 4.00 % 4.00 4.00
% %


Mgj+ (as MgCl2) 0.20 0.20 % 0.20 0.20
% %


Ca + (as CaCl2) 0.40 0.40 % 0.40 0.40
% %


Water 65.45 65 % 65.48 65.1
% %


All formulas are adjusted to pH 7.
In Examples 7 - 10, the variants recited in Tables 2 - 10, and the preferred
variants cited herein, among others, are substituted for D 79, with
substantially similar
results.
Liquid fabric cleaning compositions of the present invention are illustrated
by the
following examples.


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WO 99/49057 PCT/IB99/00520
78
Examples 11 - 13
Liquid Fabric Cleaning Compositions
Ex.ll Ex. l2 Ex. l3


O 70, 75 - 82 0.05 % 0.03 0.30
%


Sodiuam C,2 - C,4 alkyl sulfate 20.0 % 20.0 20.0
%


2-Butyl octanoic acid 5.0 % 5.0 % 5.0


Sodium citrate 1.0 % I.0 % 1.0


C,o alcohol ethoxylate (3) 13.0 % 13.0 13.0
%


Monoethanolamine 2.50 % 2.50 2.50
%


Water/propylene glycol/ethanol 58.45 58.47 58.20
(100:1:1) % %


In Examples 11 - 13, the variants recited in 'fables 2 - 10, and the preferred
variants cited herein, among others, are substituted for O 70, 75 - 82, with
substantially
similar results.
Personal Care Compositions
The present variants are particularly suited for use in personal care
compositions
such as, for example, leave-on and rinse-off hair conditioners, shampoos,
leave-on and
rinse-off acne compositions, facial milks and conditioners, shower gels,
soaps, foaming
and non-foaming facial cleansers, cosmetics, hand, facial, and body lotions
and
moisturizers, leave-on facial moisturizers, cosmetic and cleansing wipes, oral
care
compositions, and contact lens care compositions. The present personal care
compositions comprise one or more variants of the present invention and a
personal care
carrier.
To illustrate, the present variants are suitable for inclusion in the
compositions
described in the following references: U.S. Pat. No. 5,641,479, Linares et
al.. issued
June 24, 1997 (skin cleansers); U.S. Pat. No. 5,599,549, Wivell et al.. issued
February 4,
1997 (skin cleansers); U.S. Pat. No. 5,585,104, Ha et aL, issued December I7,
1996
(skin cleansers); U.S. Pat. No. 5,540,852, Kefauver et al., issued July 30,
1996 (skin
cleansers); U.S. Pat. No. 5,510,050, Dunbar et al., issued April 23, 1996
(skin cleansers);


CA 02324422 2000-09-22
WO 99/49057 PCT/IB99/00520
79
U.S. Pat. No. 5,612,324, Guang Lin et al., issued March 18, 1997 (anti-acne
preparations); U.S. Pat. No. 5,587,176, Warren et al., issued December 24,
1996 (anti-
acne preparations); U.S. Pat. No. 5,549,888, Venkateswaran, issued August 27,
1996
(anti-acne preparations); U.S. Pat. No. 5,470,884, Corless et aL, issued
November 28,
1995 (anti-acne preparations); U.S. Pat. No. 5,650,384, Gordon et al., issued
July 22,
1997 (shower gels); U.S. Pat. No. 5,607,678, Moore et al., issued March 4,
1997 (shower
gels); U.S. Pat. No. 5,624,666, Coffindaffer et al., issued April 29, 1997
(hair
conditioners and / or shampoos); U.S. Pat. No. 5,618,524, Bolich et al.,
issued April 8,
1997 (hair conditioners and / or shampoos); U.S. Pat. No. 5,612,301, Inman,
issued
March 18, 1997 (hair conditioners and / or shampoos); U.S. Pat. No. 5,573,709,
Wells,
issued November 12, 1996 {hair conditioners and / or shampoos); U.S. Pat. No.
5,482,703, Pines, issued January 9, 1996 (hair conditioners and / or
shampoos); U.S. Pat.
No. Re. 34,584, Grote et al., Reissued April 12, 1994 (hair conditioners and /
or
shampoos); U.S. Pat. No. 5,641,493, Date et al., issued June 24, 1997
(cosmetics); U.S.
Pat. No. 5,605,894, Blank et al., issued February 25, 1997 (cosmetics); U.S.
Pat. No.
5,585,090, Yoshioka et al., issued December 17, 1996 (cosmetics); U.S. Pat.
No.
4,939,179, Cheney et al., issued July 3, 1990 (hand, face, and / or body
lotions); U.S.
Pat. No. 5,607,980, McAtee et al., issued March 4, 1997 (hand, face, and / or
body
lotions); U.S. Pat. No. 4,045,364, Richter et al., issued August 30, 1977
(cosmetic and
cleansing wipes); European Patent Application, EP 0 619 074, Touchet et al.,
published
October 12, 1994 (cosmetic and cleansing wipes); U.S. Pat. No. 4,975,217,
Brown-
Skrobot et al.. issued December 4, 1990 (cosmetic and cleansing wipes); U.S.
Pat. No.
5,096,700, Seibel, issued March 17, 1992 (oral cleaning compositions); U.S.
Pat. No.
5,028,414, Sampathkumar, issued July 2, 1991 (oral cleaning compositions);
U.S. Pat.
No. 5,028,41 S, Benedict et al.. issued July 2, 1991 (oral cleaning
compositions); U.S.
Pat. No. 5,028,415, Benedict et al., issued July 2, 1991 (oral cleaning
compositions);
U.S. Pat. No. 4,863,627, Davies et al., September 5, 1989 (contact lens
cleaning
solutions); U.S. Pat. No. Re. 32,672, Huth et al, reissued May 24, 1988
(contact lens
cleaning solutions); and U.S. Pat. No. 4,609,493, Schafer, issued September 2,
1986
(contact lens cleaning solutions).


CA 02324422 2000-09-22
WO 99/49057 PCT/IB99/00520
To further illustrate oral cleaning compositions of the present invention, a
pharmaceutically-acceptable amount of one or more variants of the present
invention are
included in compositions useful for removing proteinaceous stains from teeth
or
dentures. As used herein, "oral cleaning compositions" refers to dentifrices,
toothpastes,
toothgels, toothpowders, mouthwashes, mouth sprays, mouth gels, chewing gums,
lozenges, sachets, tablets, biogels, prophylaxis pastes, dental treatment
solutions, and the
like. Preferably, the oral cleaning compositions comprise (from about 0.0001 %
to about
20% of one or more variants of the present invention, more preferably from
about
0.001 % to about 10%, more preferably still from about 0.01 % to about 5%, by
weight
of the composition, and a pharmaceutically-acceptable carrier. As used herein,
"pharmaceutically-acceptable" means that drugs, medicaments or inert
ingredients which
the term describes are suitable for use in contact with the tissues of humans
and lower
animals without undue toxicity, incompatibility, instability, irritation,
allergic response,
and the like, commensurate with a reasonable benefit / risk ratio.
Typically, the pharmaceutically-acceptable oral cleaning carrier components of
the oral cleaning components of the oral cleaning compositions will generally
comprise
from about 50% to about 99.99%, preferably from about 65% to about 99.99%,
more
preferably from about 65% to about 99%, by weight of the composition.
The pharmaceutically-acceptable Garner -components and optional components
which may be included in the oral cleaning compositions of the present
invention are
well known to those skilled in the art. A wide variety of composition types,
carrier
components and optional components useful in the oral cleaning compositions
are
disclosed in the references cited hereinabove.
In another embodiment of the present invention, denture cleaning compositions
for cleaning dentures outside of the oral cavity comprise one or more variants
of the
present invention. Such denture cleaning compositions comprise an effective
amount of
one or more of the variants, preferably from about 0.0001 % to about 50% of
one or more
of the variants, more preferably from about 0.001% to about 35%, more
preferably still
from about 0.01 % to about 20%, by weight of the composition, and a denture
cleansing
carrier. Various denture cleansing composition formats such as effervescent
tablets and


CA 02324422 2000-09-22
WO 99/49057 PCT/IB99/00520
81
the like are well known in the art see e. ., U.S. Pat. No. 5,055,305, Youn ,
and are
generally appropriate for incorporation of one or more of the variants for
removing
proteinaceous stains from dentures.
In another embodiment of the present invention, contact lens cleaning
compositions comprise one or more variants of the present invention. Such
contact lens
cleaning compositions comprise an effective amount of one or more of the
variants,
preferably from about 0.01% to about 50% of one or more of the variants, more
preferably from about 0.01 % to about 20%, more preferably still from about 1
% to
about 5%, by weight of the composition, and a contact lens cleaning carrier.
Various
contact lens cleaning composition formats such as tablets, liquids and the
like are well
known in the art and are generally appropriate for incorporation of one or
more variants
of the present invention for removing proteinaceous stains from contact
lenses.
The contact Iens cleaning composition embodiment of the present invention is
illustrated by Examples 14 - 17.
Examples 14 - 17
Contact Lens Cleaning Solution
Ex. Ex.lS Ex. l6 Ex. l7
l4


075-82 0.01 0.5% 0.1 % 2.0%
%


Glucose 50.0 50.0 50.0 50.0
% % %


Nonionic surfactant (polyoxyethlene2.0 2.0 % 2.0 % 2.0
polyoxypropylene copolymer)%


Anionic surfactant (polyoxyethylene1.0 1.0 % 1.0 % 1.0
alkylphenylether sodium %
sulfricester)


Sodium Chloride 1.0 1.0 % 1.0 % 1.0
%


Borax 0.30 0.30 0.30 0.30
% % %


Water 45.69 45.20 45.60 43.70
% % %




CA 02324422 2000-09-22
WO 99/49057 PCT/IB99/00520
82
in Examples 14 - 17, the variants recited in Tables 2 - 10, and the preferred
variants cited herein, among others, are substituted for O 75 - 82 with
substantially
similar results.
Examples 18 - 21 illustrate the use of the present variants in bodywash
products:
Examples 18 - 21
Bodywash Products
Ex. l8 Ex. l9 Ex.20 Ex.21


Water 62.62 65.72 % 57.72 % 60.72
%


Disodium EDTA 0.2 % 0.2 % 0.2 % 0.2


Glycerine 3.0 % 3.0 % 3.0 % 3.0


Polyquaternium 10 0.4 % 0.4 % 0.4 % 0.4


Sodium laureth sulphate12.0 12.0 % 12.0 % 12.0
%


Cocamide MEA 2.8 % 2.8 % 2.8 % 2.8


Sodium lauraphoacetate6.0 % 6.0 % 6.0 % 6.0


Myristic Acid 1.6 % 1.6 % 1.6 % 1.6


Magnesium sulphate 0.3 % 0.3 % 0.3 % 0.3
heptahydrate


Trihydroxystearin 0.5 % 0.5 % 0.5 % 0.5


PEG-6 caprylic / 3.0 % - - -
capric
triglycerides


Sucrose polyesters 3.0 % - - -
of
cottonate fatty
acid


Sucrose polyesters 3.0 % - 4.0 % -
of
behenate fatty acid


Petrolatum - 4.0 % 8.0 % -


Mineral Oil - - - 6.0


DMDM Hydantoin 0.08 0.08 % 0.08 % 0.08
%


D75-83,Y217L 0.1 % 2.0% 2.0% 5.0%


Citric Acid 1.40 1.40 % 1.40 % 1.40
%




CA 02324422 2000-09-22
WO 99/49057 PCT/IB99/00520
83
In Examples 18 - 21, the variants recited in Tables 2 - 1 p, and the preferred
variants cited herein, among others, are substituted for O 75 - 83, Y217L,
with
substantially similar results.
Examples 18 - 21 illustrate the use of the present variants in facewash
products:
EXAMPLES 22 - 25
Facewash Products
Ex.22 Ex.23 Ex.24 Ex.25


Water 66.52 65.17 68.47 68.72
% % %


Disodium EDTA 0.1 % 0.1 % 0.2 % 0.2


Citric Acid - - 1.4 % 1.4


Sodium Laureth-3 Sulfate 3.0 % 3.5 % - -


Sodium Laureth-4 Carboxylate3.0 % 3.5 % - -


Laureth-12 1.0 % 1.2 % - -


Polyquaternium 10 - - 0.4 % 0.4


Polyquaternium 25 0.3 % 0.3 % - -


Glycerine 3.0 % 3.0 % 3.0 % 3.0


Sodium Lauroamphoacetate - - 6.0 % 6.0


Lauric Acid 6.0 % 6.0 % 3.0 % 3.0


Myristic Acid - - 3.0 % 3.0


Magnesium sulphate heptahydrate2.3 % 2.0 % 2.0 % 2.0


Triethanol amine 4.0 % 4.0 % 4.0 % 4.0


Trihydroxystearin 0.5 % 0. S 0.5 % 0.5
%


Sucrose polyesters of 2.0 % 2.0 % -
behenate
fatty acid


Sucrose polyesters of 3.0 % 2.0 % - -
cottonate
fatty acid


PEG-6 caprylic / capric - - - 2.0
triglycerides




CA 02324422 2000-09-22
WO 99/49057 PCT/IB99/00520
84
Petrolatum - - 4.0 % -


Mineral Oil - - - 2.0


Cocamidopropyl betaine 2.0 % 3.0 % 1.8 % 1.8


Lauryl dimethylamine oxide1.0 % 1.2 % 1.2 % 1.2


Dex Panthenol 1.0 % 0.25 0.25 -
% %


DMDM Hydantoin 0.08 0.08 0.08 0.08
% % %


~ 73, I79M 1.0 % 2.0 % 0.5 % 0.5


Fragrance 0.2 % 0.2 % 0.2 % 0.2


In Examples 22 - 25, the variants recited in Tables 2 - 10, and the preferred
variants cited herein, among others, are substituted for D 73, I79M, with
substantially
similar results.
Examples 26 - 27 illustrate the use of the present variants in leave-on skin
moisturizing compositions:
EXAMPLES 26 - 27
Leave-on Skin Moisturizing Composition
Ex. 26 Ex. 27


Glycerine 5.0 % -


Stearic acid 3.0


C~ i-,3 Isoparaffin2.0 % -


Glycol stearate 1.5 % -


Propylene glycol - 3.0


Mineral oil 1.0 % 10.0


Sesame oil - 7.0


Petrolatum - 1.8


Triethanolamine 0.7 % -


Cetyl acetate 0.65 % -


Glyceryl stearate 0.48 % 2.0


TEA stearate - 2.5




CA 02324422 2000-09-22
WO 99/49057 PCT/IB99/00520
Cetyl alcohol 0.47 % -


Lanolin alcohol - 1.8


DEA - cetyl phosphate0.25 % -


Methylparaben 0.2 % 0.2


Propylparaben 0.12 % 0.1


Carbomer 934 0.11 % -


Disodium EDTA 0.1 % -


075-83 0.1% 0.5%


Water 84.32 % 71.1


In Examples 26 - 27, the variants recited in Tables 2 - 10, and the preferred
variants cited herein, among others, are substituted for O 75 - 83 with
substantially
similar results.
Example 28 illustrates the use of the present variants in cleansing wipe
commpositions:
EXAMPLE 28
Cleansing Wipe Composition
Propylene Glycol 1.0


Ammonium lauryl sulfate 0.6


Succinic acid 4.0


Sodium succinate 3.2


Triclosan~ 0.15


~ 75 - 82, G70V 0.05


Water 91.0


The above composition is impregnated onto a woven absorbent sheet comprised of
cellulose and / or polyester at about 250 %, by weight of the absorbent sheet.


CA 02324422 2000-09-22
WO 99/49057 PCT/IB99/00520
86
In Example 28, the variants recited in Tables 2 - 10, and the preferred
variants
cited herein, among others, are substituted for D 75 - 82, G70V with
substantially similar
results.


CA 02324422 2000-09-22
WO 99/49057 PCT/IB99/00520
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: RUBINGH, D.N. et al.
(ii) TITLE OF INVENTION: SERINE PROTEASE VARIANTS HAVING DECREASED
IMMUNOGENICITY
(iii) NUMBER OF SEQUENCES: 1
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: THE PROCTER & GAMBLE COMPANY
(B) STREET: 11810 EAST MIAMI RIVER ROAD
(C) CITY: ROSS
(D) STATE: OH
(E) COUNTRY: USA
(F) ZIP: 45061
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: HERSKO, BART S.
(B) REGISTRATION NUMBER: 32,572
(C) ATTORNEY DOCKET NO.
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 513-627-0633
(B) TELEFAX: 513-627-0260
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 275 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: l:
Ala Gln Ser VaI Pro Tyr Gly Val Ser Gln Ile Lys Ala Pro Ala Leu
1 5 10 15
His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp
20 25 30
Ser Gly Ile Asp Ser Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala
35 40 45


CA 02324422 2000-09-22
WO 99/.49057 PCT/IB99/00520
2
Ser Met Val Pro Ser Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His
50 55 60
Gly Thr His Val Ala Gly Thr Val Ala Ala Leu Asn Asn Ser Ile Gly
65 70 75 80
Val Leu Gly Val Ala Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu
85 90 95
Gly Ala Asp Gly Ser Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu
100 105 110
Trp Ala Ile Ala Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly
115 120 125
Pro Ser Gly Ser Ala Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala
130 135 140
Ser Gly Val Val Val Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly
195 150 155 160
Ser Ser Ser Thr Val Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala
165 170 175
Val Gly Ala Val Asp Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Val
180 185 190
Gly Pro Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln Set Thr
195 200 205
Leu Pro Gly Asn Lys Tyr Gly Ala Tyr Asn Gly Thr Ser Met AIa Ser
210 215 220
Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn
225 230 235 240
Trp Thr Asn Thr Gln Val Arg Ser Ser Leu Glu Asn Thr Thr Thr Lys
245 250 255
Leu Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala
260 265 270
Ala Ala Gln
275