Note: Descriptions are shown in the official language in which they were submitted.
CA 02290038 1999-11-10
WO 99/02989 PCT/GB98/02042
The present invention relates to chaperone polypeptides which are active in
folding or
maintaining the structural integrity of other proteins. In particular, the
present
invention relates to fragments of chaperone polypeptides which are active in
vivo and
can complement or potentiate the activity of intact molecular chaperones in
vivo, and to
methods for identifying such fragments.
Chaperones are in general known to be large multisubunit protein assemblies
essential
in mediating polypeptide chain folding in a variety of cellular compartments.
Families
of chaperones have been identified, for example the chaperonin hsp60 family
otherwise
known as the cpn60 class of proteins are expressed constitutively and there
are
examples to be found in the bacterial cytoplasm (GroEL), in endosymbiotically
derived
mitochondria (hsp60) and in chloroplasts (Rubisco binding protein}. Another
chaperone
family is designated TFSSITCPI and found in the thermophilic archaea and the
evolutionarily connected eukaryotic cytosol. A comparison of amino acid
sequence
data has shown that there is at least 50 % sequence identity between
chaperones found in
prokaryotes, mitochondria and chloroplasts (Ellis R J and Van der Vies S M
(1991)
Ann Rev Biochem ~Q: 321-347).
A typical chaperonin is GroEL which is a member of the hsp60 family of heat
shock
proteins. GroEL is a tetradecamer wherein each monomeric subunit (cpn60m) has
a
molecular weight of approximately 57kD. The tetradecamer facilitates the in
vitro
folding of a number of proteins which would otherwise misfold or aggregate and
precipitate. The structure of GroEL from E. coli has been established through
X-ray
crystallographic studies as reported by Braig K et al (1994) Nature ~1: 578-
586. The
holo protein is cylindrical, consisting of two seven-membered rings that form
a large
central cavity which according to Ellis R J and Hartl F U (1996) FASEB Journal
IQ:
20-26 is generally considered to be essential for activity. Some small
proteins have
been demonstrated to fold from their denatured states when bound to GroEL
(Gray T E
and Fersht A R (1993) J Mol Biol ~: 1197-1207; Hunt J F et al (1996) Nature
~Q:
37-45; Weissman J S et al (1996) Cell $4: 481-490; Mayhew M et al (1996)
Nature
CA 02290038 1999-11-10
WO 99/02989 PCT/GB98/02042
2
~: 420-426; Corrales F J and Fersht A R (1995) Proc Nat Acad Sci 9~: 5326-
5330)
and it has been argued that a cage-like structure is necessary to sequester
partly folded
or assembled proteins (Elks R J and Hartl F U (1996) supra.
The entire amino acid sequence of E. coli GroEL is also known (see Braig K et
al
( 1994) supra) and three domains have been ascribed to each cpn60m of the holo
chaperonin (tetradecamer). These are the intermediate (amino acid residues 1-
5, 134-
190, 377-408 and 524-548), equatorial (residues 6-133 and 409-523) and apical
(residues 191-376) domains.
International patent application W098113496 (incorporated herein by reference)
describes the generation of fragments of molecular chaperones which
surprisingly
possess molecular chaperone activity in monomeric form. A number of preferred
fragments are identified, some of which are based on the apical domain of
GroEL. One
of the most preferred fragments includes amino acid residues 191-376 of E.
coli
GroEL. However, the activity reported in W098/13496 is largely in vitro
activity.
There are no examples of in vivo assessment of chaperone activity.
It has surprisingly been found that in vitro activity of molecular chaperone
fragments
does not necessarily reflect in vivo activity of the same fragments.
Accordingly, in a
first aspect, the present invention provides a method for determining whether
a
fragment of a molecular chaperone is active in vivo, comprising the steps of:
a) providing a cell with a deficient molecular chaperone activity;
b) administering the molecular chaperone fragment to the cell;
c) determining whether the molecular chaperone fragment complements the
deficient endogenous molecular chaperone activity.
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WO 99/02989 PCT/GB98/02042
3
In a further aspect, the invention provides a method for providing a chaperone
activity
in vivo, comprising administering to a cell a fragment of a molecular
chaperone which
. has in vivo activity.
' S Advantageously, the molecular chaperone fragment complements a mutant or
depressed
endogenous molecular chaperone activity.
According to a third aspect, the invention relates to the use of a fragment of
a
molecular chaperone to complement a mutant or deficient molecular chaperone
activity
in vivo.
Fig. 1. Schematic representation of the plasmid construction and organisation.
amp,
~i-lactamase; cm, chloramphenicol acetyl transferase; R, resistance; rbs,
ribosome
binding site; plo, promoter/operator: tet, tetracycline. Relevant restriction
sites are
indicated.
Fig. 2. Three-dimensional structure of minichaperone GroEL(191-345) solved at
2.5 A
(Zahn, R., Buckle, A. M., Perret, S., Johnson, C. M. J., Corrales, F. J.,
Golbik, R. &
Fersht, A. R. (1996) Proc. Natl. Acad. Sci. USA 93, 15024-15029). Secondary
structure representation is drawn with MolScript (Kraulis, P. (1991) J. Appl.
Crystallogr. 24, 946-950). Positions mentioned in the text are indicated
{residues
numbered as in Hemmingsen et al. , (1988) Nature 333, 330-334).
Fig. 3. In vitro refolding of rhodanese in the presence of GroEL
minichaperones.
Relative enzymatic activity of rhodanese (0.1 ~.M) after refolding in the
presence (+)
or absence (-) of GroEL (2.5 ~M monomer), GroES (2.5 ~cM monomer), ATP (2 mM),
sht-GroEL191-376 (2.5 uM), sht-GroEL191-345 (2.5 ~cM), sht-GroEL193-335 wild-
type and mutant Y203E (2.5 ~,M), or bovine serum albumin (BSA; 45 uglml), from
8
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WO 99/02989 PCTIGB98/02042
4
M urea (U). The yield of refolding activity was measured after 15 min at
25°C. 100%
activity was obtained with native rhodanese (N). Standard error bars are
shown.
Fig. 4. Suppression of the temperature-sensitive phenotypes of E. coli SV2
(groEL4~
and SV6 (groEL673) strains by over-expression of GroEL or minichaperones. The
vectors are indicated on the right-hand side.
Fig. 5. Supplementation of low levels of GroEL in E. coli by minichaperones.
E. toll
AI90[pBAD-ELJ has the chromosomal groEL gene deleted by P1 transduction (Ivic,
A., Olden, D., Wallington, E. J. & Lund, P. A. (1997) Gene 194, 1-8) and
essential
GroEL is provided by a plasmid-borne copy of the gene pBAD-EL, which can be
tightly regulated by arabinose (Guzman, L.-M., Belin, D., Carson, M. J. &
Beckwith,
J. (1995) J. Bacteriol. 177, 4121-4130). In the absence of arabinose, the
cells are not
viable, unless pJCGroEL(1-548) is present, which expresses intact GroEL (O).
pJCsht
(D) and pJCGroELsht-(193-335)Y203E (~) plasmids are controls, and cells
containing
these show increased viability with increasing arabinose concentrations. Over-
expression of sht-GroEL(191-376) (~) marginally increases viability; sht-
GroEL(191-
345) (~) and (193-335) (i) minichaperones both give significant increases.
Each value
recorded is the plating efficiency relative to that in the presence of 0.2%
arabinose
(100%).
Fig. 6. Effect of over-expressing GroEL or the minichaperones on Lorist6
replication.
Cultures of TG1 carrying Lorist6 and one of the pBAD30 series were , -tred on
LB
plates containing kanamycin and ampicillin in presence or absence of 0.2 %
arabinose
at 37°C. The percentage of cells forming kanamycin resistant colonies
in presence of
arabinose compared to the number formed in the absence of arabinose ( 100 % )
is shown
for each of the pBAD30 series of expression vectors. Lorist6 plasmid encodes
resistance to kanamycin and uses the bacteriophage ~, origin of replication
(Gibson, T.
J., Rosenthal, A. & Waterston, R. H. (1987) Gene 53, 283-286). Arabinose
induces
expression of the pBAD30 series vectors; loss of kanatnycin resistance
reflects
inhibition of Lorist6 replication.
CA 02290038 1999-11-10
WO 99/02989 PCTIGB98/02042
5 In accordance with the present invention, it has surprisingly been found
that although
various fragments of molecular chaperones are capable of promoting the folding
of
polypeptides in vitro, this does not necessarily mean that they are capable of
providing
some or all of the functions of endogenous molecular chaperones in vivo. This
conclusion is of great importance, because in many therapeutic applications
envisaged
for molecular chaperones, which involve the correction of defects in protein
folding, an
in vivo activity is required. Thus, the present invention provides a method
for
identifying fragments of molecular chaperones which are potentially useful in
in vivo
applications, as well as methods for using such fragments in vivo.
A further surprising finding of the present invention is that fragments of
molecular
chaperones are able to replace specific activities of molecular chaperones in
vivo, but
often not able to replace the chaperone activity in its entirety. This means
that, where
the molecular chaperone is essential for cell viability, it is not possible to
test the
activity of chaperone fragments by deleting the endogenous chaperone activity
and
attempting to rescue it. Thus, the invention provides for the use of
deficiencies in
molecular chaperones in order to screen chaperone fragments for the ability to
complement these deficiencies.
In the present text, references to the singular, for example as in "a cell",
are to be
interpreted as references to the singular both in isolation and when
encompassed in a
plurality. Thus, terms such as, for example, "a cell" and "one or more cells"
and "at
least one cell" are equivalent.
As used herein, a fragment of a molecular chaperone is any fragment of a
molecular
chaperone polypeptide which has molecular chaperone activity. Molecular
chaperones
are well known in the art, several families thereof being characterised. The
invention
CA 02290038 1999-11-10
WO 99/02989 PCT/GB98/02042
6
is applicable to any molecular chaperone molecule, which term includes, for
example,
the molecular chaperones selected from the following non-exhaustive Group:
p90 Calnexin Salopek et al., J. Investig Dermato! Symp Proc (1996)
1:195
HSP family Walsh et al. , Cell Mol. Life Sci.
(1997) 53: i98
HSP 70 family Rokutan et al., J. Med. Invest. (1998)
44:137
DNA K Rudiger et al., Nat. Struct. Bioi.
(1997) 4:342
DNAJ Cheetham et al., Cell Stress Chaperones
(1998) 3:28
HSP 60 family; GroELRichardson et al., Trends Biochem.
(1998) 23:138
ER-associated chaperonesKim et al. , Endocr Rev ( 1998) 19:173
HSP 90 Smith, Biol. Chem. (1988) 379:283
Hsc 70 Hohfeld, Biol. Chem. (1988) 379:269
sHsps; SecA; SecB Beissinger et al., Biol. Chem. (1988)
379:245
Trigger factor Wang et al., FEBS Lett. (1998) 425:382
zebrafish hsp 47, Krone et al., Biochem. Cell Biol.
70 and (1997) 75:487
90
HSP 47 Nagata, Matrix Biol. (1998) 16:379
GRP 94 Nicchitta et al., Curr. Opin. Immunol. (1998) 10:103
Cpn 10 Cavanagh, Rev. Reprod. (1996) 1:28
BiP Sommer et al. , FASEB J. (1997) 11:1227
GRP 78 Brostrom et al. , Prog. Nucl. Acid. res. Mol. Biol. (1998)
58:79
Clp, FtsH Suzuki et al., Trends Biochem. Sci. (1997) 22:118
Ig invariant chain Weenink et al. Immunol. Cell biol. (1997) 75:69
mitochondria! hsp 70 Horst et al., BBA (1997) 1318:71
EBP Hinek, Arch. Immunol. Ther. Exp.
(1997) 45:15
mitochondrial Langer et al., Experientia (1996)
m-AAA 52:1069
Yeast Ydj 1 Lyman et al. , Experientia ( 1996)
52:1042
Hsp 104 Tuite et al. , Trends Genet. (1996)
12:467
ApoE Blain et al. , Presse Med. ( 1996)
25:763
CA 02290038 1999-11-10
WO 99/02989 PCT/GB98/02042
7
Syc Wattiau et al., Mol. Microbiol. (1996) 20:255
Hip Ziegelhoffer et al., Curr. Biol. (1996) 6:272
TriC family Hendrick et al. , FASEB J. ( 1995) 9:1559
CCT Kubota et al., Eur. J. Biochem. (1995) 230:3
PapD, calmodulin Stanfield et al., Curr. Opin. Struct. Biol. (1995) 5:103
Preferably, the molecular chaperone is a chaperone of the hsp60 class, such as
the E.
toll chaperone GroEL.
The method of the invention allows molecular chaperone fragments active in
vivo to be
identified empirically. Accordingly, in one embodiment, fragments of molecular
chaperones may be generated randomly and tested by the method of the invention
to
assess in vivo activity.
The size of molecular chaperone fragments does not necessarily correlate with
in vivo
activity. Thus, the fragments used in the method of the invention may be
substantially
any size. Preferably, the minimum size of suitable fragments can be determined
experimentally, for example by generating crystal structures of the fragments.
residues
which display no electron density, and are thus outside the folded structure
of the
chaperone fragment, are less likely to be essential for chaperone activity.
Conversely,
fragments which do not possess enough sequence to form a stable folded
structure at all
are unlikely to be active as chaperones.
Preferably, fragments selected far analysis are of the order of between about
100 to 500
amino acids in size, advantageously about 120 to 400, more advantageously
about 130
to 300 and most advantageously about 145 to 200 amino acids in length.
Preferably, however, fragments of hsp60 class molecular chaperones may be
generated
as set forth in W098/13496. In particular, molecular chaperone fragments may
be
taken from, or comprise, residues 191-376 of GroEL or the equivalent amino
acid
positions in other hsp60 molecules.
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8
Especially preferred are fragments 191-345 and 193-335 of GroEL, or
equivalents
thereof. 193-335 is the smallest GroEL fragment which has been demonstrated to
possess chaperone activity in vitro. Other fragments, including 191-345 and
191-376,
possess in vitro chaperone activity. However, it has surprisingly been shown
that
although 191-376 has only very limited activity in vivo and is not able to
significantly
complement a deficiency in molecular chaperone activity, both of the smaller
fragments
191-345 and 193-335 are active in vivo and can complement a deficiency in
molecular
chaperone activity.
The present invention is concerned with in vivo molecular chaperone activity.
As used
herein, in vivo describes an activity which takes place inside a living cell.
It is not
necessarily intended to refer to activity in live animals, but also to include
activity in
tissue and cell culture. "Activity" is any activity which is ascribable to a
molecular
chaperone. Typically, these activities include the facilitation of protein
folding, the
latter term including the folding of unfolded proteins and the maintenance of
folded
proteins in a correctly folded conformation. Other activities of molecular
chaperones
which are included are antiviral activities, protein transport activities and
the like.
Preferably, in vivo activity is measured as the ability to rescue cell
viability when
chaperone activity is deficient. Molecular chaperones of the hsp60 such as
GroEL are
essential, and if they are ablated from a cell the cell will die. However,
mutants may
be designed in which the endogenous holochaperone is mutated, for example such
that
it is temperature sensitive. In this case, the holochaperone will be deficient
at non-
permissive temperatures. Alternatively, a cell may be transfected wish a
vector
encoding the holochaperone or a fully functional variant thereof under the
control of
regulatable sequences, such that in response to the appropriate stimuli the
chaperone
will be produced at deficient levels. In systems such as these, cell growth is
compromised in the presence of chaperone deficiency . The in vivo activity of
a
chaperone fragment may be defined as the ability of the fragment to restore
all or part
of the chaperone activity to such cells, when the fragment is introduced
therein.
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A "deficient" chaperone activity, as referred to herein. is a chaperone
activity which is
. not equal to that of a wild type molecular chaperone. For example, it may be
quantitatively deficient, such that an insufficient amount of chaperone
protein is
' S available in the cell. Alternatively, it may be qualitatively deficient,
such that the
chaperone is not able to mediate certain reactions, or cannot do so at the
rate at which a
wild type chaperone mediates them. Typically, when referring to a deficient
chaperone
activity, it is intended to refer to a deficiency in endogenous holochaperone
activity,
such as a deficiency in endogenous GroEL activity in E. coli. However, the
invention
also allows for the endogenous activity to be replaced or augmented by an
exogenous
activity, which may then be rendered deficient. For example, the endogenous
chaperone activity may be deleted and "rescued" with a regulatable chaperone
activity
encoded on a plasmid, which is susceptible to dowregulation to a deficient
state.
The cell may have a permanently deficient chaperone activity, in which case
the
deficiency is preferably not lethal, allowing the cells to grow in the absence
of a
functional chaperone fragment. Alternatively, the deficiency may be
regulatable, for
example by means of a temperature-sensitive chaperone mutant or the
introduction of a
regulatable chaperone coding sequence as set forth above.
The chaperone fragment need not be able to replace or complement all of the
lost
molecular chaperone activities. Indeed, it is believed that chaperone
fragments such as
GroEL 193-335 are only capable of complementing certain protein folding
activities of
holo GroEL in vivo. Whilst not wishing to be bound by theory, it is possible
that the
complementing of these activities is itself sufficient to restore partial cell
growth to a
cell deficient in holo GroEL, for example by allowing bolo GroEL to be
diverted to
other processes where the 193-335 fragment cannot replace it.
Cells suitable for practising the present invention may be of any suitable
type. Host
cells such as prokaryote, yeast and higher eukaryote cells may be used.
Suitable
prokaryotes include eubacteria, such as Gram-negative or Gram-positive
organisms,
CA 02290038 1999-11-10
WO 99/02989 PCT/GB98/02042
such as E. coli, e.g. E. coli K-I2 strains, DHSa and HB101, or Bacilli.
Further hosts
suitable for chaperone fragment encoding vectors include eukaryotic microbes
such as
filamentous fungi or yeast, e.g. Saccharomyces cerevisiae. Higher eukaryotic
cells
include insect and vertebrate cells, particularly mammalian cells, including
human cells,
5 or nucleated cells from other multicellular organisms.
The propagation of vertebrate cells in culture (tissue culture) is a routine
procedure.
Examples of useful mammalian host cell lines are epithelial or fibroblastic
cell lines
such as Chinese hamster ovary (CHO) cells, NIH 3T3 cells, HeLa cells or 293T
cells.
10 The host cells referred to in this disclosure comprise cells in in vitro
culture as well as
cells that are within a host animal.
Host cells are transfected with vectors according to the invention and
cultured in
conventional nutrient media modified as appropriate for inducing promoters,
selecting
IS transformants, or amplifying the genes encoding the desired sequences.
Heterologous
DNA may be introduced into host cells by any method known in the art, such as
transfection with a vector encoding a heterologous DNA by the calcium
phosphate
coprecipitation technique or by electroporation. Numerous methods of
transfection are
known to the skilled worker in the field. Successful transfection is generally
recognised
when any indication of the operation of this vector occurs in the host cell.
Transformation is achieved using standard techniques appropriate to the
particular host
cells used.
incorporation of cloned DNA into a suitable expression vector, transfection of
eukaryotic cells with a piasmid vector or a combination of plasmid vectors,
each
encoding one or more distinct genes or with linear DNA, and selection of
transfected
cells are well known in the art (see, e.g. Sambrook et aI. (1989) Molecular
Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press).
Transfected or transformed cells are cultured using media and culturing
methods known
in the art, preferably under conditions, whereby chaperone fragment encoded by
the
CA 02290038 1999-11-10
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11
DNA is expressed. The composition of suitable media is known to those in the
art, so
that they can be readily prepared. Suitable culturing media are also
commercially
available.
In accordance with the present invention, the molecular chaperone fragment
being
tested is administered to the test cell, for example, by administration of the
fragment in
polypeptide form, or by introduction into the cell of a nucleic acid encoding
the
fragment such that it is expressed in the cell. Insertion of nucleic acid
sequences, by
processes such as transfection, transduction, electroporation of otherwise, is
preferred.
la
Preferably, nucleic acid encoding a molecular chaperone fragment is introduced
by
means of a vector.
As used herein, vector (or plasmid} refers to discrete elements that are used
to
introduce heterologous DNA into cells for expression thereof. Selection and
use of such
vehicles are well within the skill of the artisan. Many vectors are available,
and
selection of appropriate vector will depend on, for example, the size of the
DNA to be
inserted into the vector, and the host cell to be transformed with the vector.
Each vector
contains various components depending on its function and the host cell for
which it is
compatible. The vector components generally include, but are not limited to,
one or
more of the following: an origin of replication, one or more marker genes, an
enhancer
element, a promoter, a transcription termination sequence and a signal
sequence.
Both expression and cloning vectors generally contain nucleic acid sequences
that
enable the vector to replicate in one or more selected host cells. Typically
in cloning
vectors, this sequence is one that enables the vector to replicate
independently of the
host chromosomal DNA, and includes origins of replication or autonomously
replicating sequences. Such sequences are well known for a variety of
bacteria, yeast
and viruses. The origin of replication from the plasmid pBR322 is suitable for
most
Gram-negative bacteria, the 2~ plasmid origin is suitable for yeast, and
various viral
origins (e.g. SV 40, polyoma, adenovirus) are useful for cloning vectors in
mammalian
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12
cells. Generally, the origin of replication component is not needed for
mammalian
expression vectors unless these are used in mammalian cells competent for high
level
DNA replication, such as COS cells.
Most expression vectors are shuttle vectors, i.e. they are capable of
replication in at
least one class of organisms but can be transfected into another class of
organisms for
expression. For example, a vector may be cloned in E. coli and then the same
vector
may be transfected into yeast or mammalian cells even though it is not capable
of
replicating independently of the host cell chromosome. DNA may also be
amplified by
PCR and may be directly transfected into the host cells without any
replication
component.
Advantageously, a vector may contain a selection gene also referred to as
selectable
marker. This gene encodes a protein necessary for the survival or growth of
transformed host cells grown in a selective culture medium. Host cells not
transformed
with the vector containing the selection gene will not survive in the culture
medium.
Typical selection genes encode proteins that confer resistance to antibiotics
and other
toxins, e.g. ampicillin, neomycin, methotrexate or tetracycline, complement
auxotrophic deficiencies, or supply critical nutrients not available from
complex media.
As to a selective gene marker appropriate for yeast, any marker gene can be
used which
facilitates the selection for transformants due to the phenotypic expression
of the
marker gene. Suitable markers for yeast are, for example, those conferring
resistance to
antibiotics 6418, hygromycin or bleomycin, or provide for prototrophy in an
auxotrophic yeast mutant, for example the URA3, LEU2, LYS2, TRP1, or HIS3
gene.
Since the replication of vectors is conveniently done in E. coli, an E. toll
genetic
marker and an E. toll origin of replication are advantageously included. These
can be
obtained from E. toll plasmids, such as pBR322, Bluescript~ vector or a pUC
plasmid,
e.g. pUCl8 or pUCl9, which contain both E. toll replication origin and E. toll
genetic
marker conferring resistance to antibiotics, such as ampicillin.
CA 02290038 1999-11-10
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Suitable selectable markers for mammalian cells are those that enable the
identification
of cells competent to take up chaperone fragment nucleic acid, such as
dihydrofolate
reductase (DHFR, methotrexate resistance), thymidine kinase, or genes
conferring
resistance to 6418 or hygromycin. The mammalian cell transformants are placed
under
selection pressure which only those transformants which have taken up and are
expressing the marker are uniquely adapted to survive. In the case of a DHFR
or
glutamine synthase (GS) marker, selection pressure can be imposed by culturing
the
transformants under conditions in which the pressure is progressively
increased,
thereby leading to amplification (at its chromosomal integration site) of both
the
selection gene and the linked DNA that encodes chaperone fragment.
Amplification is
the process by which genes in greater demand for the production of a protein
critical
for growth, together with closely associated genes which may encode a desired
protein,
are reiterated in tandem within the chromosomes of recombinant cells.
Increased
quantities of desired protein are usually synthesised from thus amplified DNA.
Expression vectors usually contain a promoter that is recognised by the host
organism
and is operably linked to chaperone fragment nucleic acid. Such a promoter may
be
inducible or constitutive. The promoters are operably linked to DNA encoding
chaperone fragment by removing the promoter from the source DNA by restriction
enzyme digestion and inserting the isolated promoter sequence into the vector.
Both the
native chaperone fragment promoter sequence and many heterologous promoters
may
be used to direct amplification and/or expression of chaperone fragment DNA.
The
term "operably linked" refers to a juxtaposition wherein the components
described are
in a relationship permitting them to function in their intended manner. A
control
sequence "operably linked" to a coding sequence is ligated in such a way that
expression of the coding sequence is achieved under conditions compatible with
the
control sequences.
Promoters suitable for use with prokaryotic hosts include, for example, the ø-
lactamase
and lactose promoter systems, alkaline phosphatase, the tryptophan (trp)
promoter
CA 02290038 1999-11-10
WO 99/02989 PCT/GB98102042
14
system and hybrid promoters such as the tic promoter. Their nucleotide
sequences have
been published, thereby enabling the skilled worker operably to ligate them to
DNA
encoding chaperone fragment, using linkers or adaptors to supply any required
restriction sites. Promoters for use in bacterial systems will also generally
contain a
S Shine-Delgarno sequence operably linked to the DNA encoding chaperone
fragment.
Preferred expression vectors are bacterial expression vectors which comprise a
promoter of a bacteriophage such as phagex or T7 which is capable of
functioning in
the bacteria. In one of the most widely used expression systems, the nucleic
acid
encoding the fusion protein may be transcribed from the vector by T7 RNA
polymerise
(Studier et al, Methods in Enzymol. 185; 60-89, 1990). In the E. coli
BL21(DE3) host
strain, used in conjunction with pET vectors, the T7 RNA polymerise is
produced from
the ~.-lysogen DE3 in the host bacterium, and its expression is under the
control of the
IPTG inducible lac UVS promoter. This system has been employed successfully
for
over-production of many proteins. tmternauveiy the potyn~cra~c ~G11G flay
introduced on a lambda phage by infection with an int- phage such as the CE6
phage
which is commercially available (Novagen, Madison, USA). other vectors include
vectors containing the lambda PL promoter such as PLEX (Invitrogen, NL) ,
vectors
containing the trc promoters such as pTrcHisXpressTm (Invitrogen) or pTrc99
(Pharmacia Biotech, SE) , or vectors containing the tic promoter such as
pKK223-3
(Pharmacia Biotech) or PMAL (new England Biolabs, MA, USA).
Suitable promoting sequences for use with yeast hosts may be regulated or
constitutme
and are preferably derived from a highly expressed yeast gene, especially a
Saccharomyces cerevisiae gene. Thus, the promoter of the TRP1 gene, the ADHI
or
ADHII gene, the acid phosphatase (PHOS) gene, a promoter of the yeast mating
pheromone genes coding for the a- or a-factor or a promoter derived from a
gene
encoding a glycolytic enzyme such as the promoter of the enolase,
glyceraldehyde-3-
phosphate dehydrogenase (GAP), 3-phospho glycerate kinase (PGK), hexokinase,
pyruvate decaruoxyiase, pnUS~uvmus.LUmua~~, YJI4bVJlr-V-FJllvJ~Jm4~~ ~~~~----
"'-
phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase,
phosphoglucose
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isomerase or glucokinase genes. the S. cerevisiae GAL 4 gene, the S. pombe nmt
1
gene or a promoter from the TATA binding protein (TBP) gene can be used.
Furthermore, it is possible to use hybrid promoters comprising upstream
activation
sequences (UAS) of one yeast gene and downstream promoter elements including a
5 functional TATA box of another yeast gene, for example a hybrid promoter
including
the UAS(s) of the yeast PHOS gene and downstream promoter elements including a
functional TATA box of the yeast GAP gene (PHOS-GAP hybrid promoter). A
suitable
constitutive PHOS promoter is e.g. a shortened acid phosphatase PHOS promoter
devoid of the upstream regulatory elements (UAS) such as the PHOS (-173)
promoter
10 element starting at nucleotide -173 and ending at nucleotide -9 of the PHOS
gene.
Chaperone fragment gene transcription from vectors in mammalian hosts may be
controlled by promoters derived from the genomes of viruses such as polyoma
virus,
adenovirus, fowlpox virus, bovine papilioma virus, avian sarcoma virus,
15 cytomegalovirus (CMV), a retrovirus and Simian Virus 40 (SV40), from
heterologous
mammalian promoters such as the actin promoter or a very strong promoter, e.g.
a
ribosomal protein promoter, and from the promoter normally associated with
chaperone
fragment sequence, provided such promoters are compatible with the host cell
systems.
Transcription of a DNA encoding chaperone fragment by higher eukaryotes may be
increased by inserting an enhancer sequence into the vector. Enhancers are
relatively
orientation and position independent. Many enhancer sequences are known from
mammalian genes (e.g. elastase and globin). However, typically one will employ
an
enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on
the late
side of the replication origin (bp 100-270) and the CMV early promoter
enhancer. The
enhancer may be spliced into the vector at a position 5' or 3' to chaperone
fragment
DNA, but is preferably located at a site 5' from the promoter.
Advantageously, a eukaryotic expression vector encoding chaperone fragment may
comprise a locus control region (LCR). LCRs are capable of directing high-
level
integration site independent expression of transgenes integrated into host
cell
CA 02290038 1999-11-10
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16
chromatin, which is of importance especially where the chaperone fragment gene
is to
be expressed in the context of a permanently-transfected eukaryotic cell line
in which
chromosomal integration of the vector has occurred, in vectors designed for
gene
therapy applications or in transgenic animals.
Eukaryotic expression vectors may also contain sequences necessary for the
termination
of transcription and for stabilising the mRNA. Such sequences are commonly
available
from the 5' and 3' untranslated regions of eukaryotic or viral DNAs or cDNAs.
These
regions contain nucleotide segments transcribed as polyadenylated fragments in
the
untranslated portion of the mRNA encoding chaperone fragment.
An expression vector includes any vector capable of expressing chaperone
fragment
nucleic acids that are operatively linked with regulatory sequences, such as
promoter
regions, that are capable of expression of such DNAs. Thus, an expression
vector
refers to a recombinant DNA or RNA construct, such as a plasmid, a phage,
recombinant virus or other vector, that upon introduction into an appropriate
host cell,
results in expression of the cloned DNA. Appropriate expression vectors are
well
known to those with ordinary skill in the art and include those that are
replicable in
eukaryotic and/or prokaryotic cells and those that remain episomal or those
which
integrate into the host cell genome. For example, DNAs encoding chaperone
fragment
may be inserted into a vector suitable for expression of cDNAs in mammalian
cells,
e.g. a CMV enhancer-based vector such as pEVRF (Matthias, et al., (1989) NAR
17,
6418).
Particularly useful for practising the present invention are expression
vectors that
provide for the transient expression of DNA encoding chaperone fragment in
mammalian cells. Transient expression usually involves the use of an
expression vector
that is able to replicate efficiently in a host cell, such that the host cell
accumulates
many copies of the expression vector, and, in turn, synthesises high levels of
chaperone
fragment. For the purposes of the present invention, transient expression
systems are
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17
useful e.g. for identifying chaperone fragment mutants, to identify potential
phosphorylation sites. or to characterise functional domains of the protein.
Construction of vectors according to the invention employs conventional
ligation
techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and
religated in
the form desired to generate the plasmids required. If desired, analysis to
confirm
correct sequences in the constructed plasmids is performed in a known fashion.
Suitable
methods for constructing expression vectors, preparing in vitro transcripts,
introducing
DNA into host cells, and performing analyses for assessing chaperone fragment
expression and function are known to those skilled in the art. Gene presence,
amplification andlor expression may be measured in a sample directly, for
example, by
conventional Southern blotting, Northern blotting to quantitate the
transcription of
mRNA, dot blotting (DNA or RNA analysis), or in situ hybridisation, using an
appropriately labelled probe which may be based on a sequence provided herein.
Those
skilled in the art will readily envisage how these methods may be modified, if
desired.
The invention may be applied to the screening of molecular chaperone fragments
for in
vivo activity in a variety of ways. In general, a screening system requires a
read-out of
the activity of the agent being tested, and in the case of molecular
chaperones having a
general activity, cell viability or cell growth is affected by the chaperones
and is easily
monitored. In the case of more specific chaperones, however, it may be
necessary to
provide, in the cell, a reporter system whose activity is linked to the
activity of the
chaperone. For example, if the molecular chaperone in question facilitates the
folding
of a particular protein, a reporter system responsive to the protein in its
correctly folded
state may be used. For instance, where the protein is a transcription factor
or can be
incorporated into a chirneric transcription factor, a reporter system which is
transactivatable by a transcription factor may be used.
Suitable reporter systems include those based on easily detectable gene
products, such
as luciferase, as (3 galactosidase or chloramphenicol acetyltransferase (CAT).
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In the context of a screening system, the invention may be configured, for
example,
such that the transfected chaperone fragment is detected if it can rescue cell
growth in a
cell with a deficient endogenous chaperone activity. For example, growth of E.
coli is
dependent on GroEL activity. Temperature sensitive GroEL mutants will prevent
E.
coli from growing at non-permissive temperatures. Thus, E. coli may be
transfected
with vectors encoding chaperone fragments and assessed for growth at a non-
permissive
temperature, which is indicative of in vivo chaperone fragment activity.
Alternatively, a specialised chaperone required for the correct folding of a
chosen
polypeptide may be rendered deficient in the cell. In such a case, in vivo
activity of a
chaperone fragment may be assessed by measuring the biological activity of the
chosen
polypeptide, or by using a reporter system dependent on the chosen
polypeptide.
Alternatively, where the chaperone has an observed activity such as an
antiviral
activity, for example in the case of GroEL which inhibits phage ~,
replication, a
reporter system may be based on a viral promoter or replication origin.
The use of different reporter systems is likely to identify different in vivo
activities of
chaperone fragments. For example, in the case of GroEL, it is found that 193-
335
complements mutants which have very low levels of endogenous GroEL> whilst 191-
345 complements temperature sensitive mutants of GroEL. Fragment 191-376 is
able
to prevent ghage ~ replication. Thus, in a preferred aspect of the invention,
the assay
used to detect in vivo chaperone activity should reflect the use to which it
is desired to
put a minichaperone.
Endogenous chaperone proteins may be rendered deficient by a variety of means,
including deletion of genes by homologous recombination, use of antisense
molecules
or specific inhibitors. The practice of these techniques is known in the art.
Preferred,
however, is the use of homologous recombination to disrupt endogenous genes.
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i9
In a preferred aspect, the invention may be applied not only to fragments of
molecular
chaperones, but also to mutants and variants of such fragments. The variants
provided
by the present invention include splice variants encoded by mRNA generated by
alternative splicing of a primary transcript, amino acid mutants,
giycosylation variants
and other covalent derivatives of the molecular chaperone which retain the
physiological and/or physical properties thereof. Exemplary derivatives
include
molecules wherein the chaperone polypeptide is covalentiy modified by
substitution,
chemical, enzymatic, or other appropriate means with a moiety other than a
naturally
occurring amino acid. Such a moiety may be a detectable moiety such as an
enzyme or
a radioisotope. Further included are naturally occurring variants of molecular
chaperone found within a particular species, preferably a mammal. Such a
variant may
be encoded by a related gene of the same gene family, by an allelic variant of
a
particular gene, or represent an alternative splicing variant of a molecular
chaperone
gene.
Variants of the molecular chaperone fragments also comprise mutants thereof,
which
may contain amino acid deletions, additions or substitutions, subject to the
requirement
to maintain the activity of the molecular chaperone described herein. Thus,
conservative amino acid substitutions may be made substantially without
altering the
nature of the molecular chaperone, as may truncations from the 5' or 3' ends.
Deletions and substitutions may moreover be made to the fragments of the
molecular
chaperone comprised by the invention. molecular chaperone mutants may be
produced
from a DNA encoding the molecular chaperone which has been subjected to in
vitro
mutagenesis resulting e.g. in an addition, exchange andlor deletion of one or
more
amino acids. For example, substitutional, deletional or insertional variants
of the
molecular chaperone can be prepared by recombinant methods and screened for
immuno-crossreactivity with the native forms of the molecular chaperone.
The fragments, mutants and other derivatives of the molecular chaperone
preferably
retain substantial homology with the molecular chaperone . As used herein,
"homology" means that the two entities share sufficient characteristics for
the skilled
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person to determine that they are similar in origin and function. Preferably,
homology
is used to refer to sequence identity.
"Substantial homology", where homology indicates sequence identity, means more
than
5 40 % sequence identity, preferably more than 45 % sequence identity and most
preferably a sequence identity of 50% or more, as judged by direct sequence
alignment
and comparison.
Sequence homology (or identity) may moreover be determined using any suitable
10 homology algorithm, using for example default parameters. Advantageously,
the
BLAST algorithm is employed, with parameters set to default values. The BLAST
algorithm is described in detail at http:Ilwww.ncbi.nih.gov/BLAST/blast
help.html,
which is incorporated herein by reference. The search parameters are defined
as
follows, and are advantageously set to the defined default parameters.
Advantageously, "substantial homology" when assessed by BLAST equates to
sequences which match with an EXPECT value of at least about 7, preferably at
least
about 9 and most preferably 10 or more. The default threshold for EXPECT in
BLAST
searching is usually 10.
BLAST (Basic Local Alignment Search Tool) is the heuristic search algorithm
employed by the programs blastp, blastn, blastx, tblastn, and tblastx; these
programs
ascribe significance to their findings using the statistical methods of Karlin
and Altschul
(see http:/lwww.ncbi.nih.govIBLAST/blast help.html) with a few enhancements.
The
BLAST programs were tailored for sequence similarity searching, for example to
identify homologues to a query sequence. The programs are not generally useful
for
motif style searching. For a discussion of basic issues in similarity
searching of
sequence databases, see Altschul et aI. (I994) Nature Genetics 6:119-129.
The frve BLAST programs available at http:/lwww.ncbi.nlm.nih.gov perform the
following tasks:
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21
blastp compares an amino acid query sequence against a protein sequence
database;
blastn compares a nucleotide query sequence against a nucleotide sequence
database;
blastx compares the six-frame conceptual translation products of a nucleotide
query
sequence (both strands) against a protein sequence database;
tblastn compares a protein query sequence against a nucleotide sequence
database
dynamically translated in all six reading frames (both strands).
tblastx compares the six-frame translations of a nucleotide query sequence
against the
six-frame translations of a nucleotide sequence database.
BLAST uses the following search parameters:
HISTOGRAM Display a histogram of scores for each search; default is yes. (See
parameter H in the BLAST Manual).
DESCRIPTIONS Restricts the number of short descriptions of matching sequences
reported to the number specified; default limit is 100 descriptions. (See
parameter V in
the manual page). See also EXPECT and CUTOFF.
ALIGNMENTS Restricts database sequences to the number specified for which high-
scoring segment pairs (HSPs) are reported; the default limit is 50. If more
database
sequences than this happen to satisfy the statistical significance threshold
for reporting
(see EXPECT and CUTOFF below), only the matches ascribed the greatest
statistical
significance are reported. (See parameter B in the BLAST Manual).
EXPECT The statistical significance threshold for reporting matches against
database
sequences; the default value is I0, such that 10 matches are expected to be
found
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22
merely by chance, according to the stochastic model of Karlin and Altschul
(1990). If
the statistical significance ascribed to a match is greater than the EXPECT
threshold,
the match will not be reported. Lower EXPECT thresholds are more stringent,
leading
to fewer chance matches being reported. Fractional values are acceptable. (See
parameter E in the BLAST Manual).
CUTOFF Cutoff score for reporting high-scoring segment pairs. The default
value is
calculated from the EXPECT value (see above). HSPs are reported for a database
sequence only if the statistical significance ascribed to them is at Ieast as
high as would
be ascribed to a lone HSP having a score equal to the CUTOFF value. Higher
CUTOFF values are more stringent, leading to fewer chance matches being
reported.
(See parameter S in the BLAST Manual). Typically, significance thresholds can
be
more intuitively managed using EXPECT.
MATRIX Specify an alternate scoring matrix for BLASTP, BLASTX, TBLASTN and
TBLASTX. The default matrix is BLOSUM62 (Henikoff & Henikoff, 1992). The valid
alternative choices include: PAM40, PAM120, PAM250 and IDENTITY. No alternate
scoring matrices are available for BLASTN; specifying the MATRIX directive in
BLASTN requests returns an error response.
STRAND Restrict a TBLASTN search to just the top or bottom strand of the
database
sequences; or restrict a BLASTN, BLASTX or TBLASTX search to just reading
frames on the top or bottom strand of the query sequence.
FILTER Mask off segments of the query sequence that have Iow compositional
complexity, as determined by the SEG program of Wootton & Federhen (1993)
Computers and Chemistry 17:149-i63, or segments consisting of short-
periodicity
internal repeats, as determined by the XNU program of Claverie & States (1993)
Computers and Chemistry 17:191-201, or, for BLASTN, by the DUST program of
Tatusov and Lipman (see http:l/www.ncbi.nlm.nih.gov). Filtering can eliminate
statistically significant but biologically uninteresting reports from the
blast output (e.g.,
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23
hits against common acidic-, basic- or proline-rich regions), leaving the more
biologically interesting regions of the query sequence available for specific
matching
against database sequences.
' S Low complexity sequence found by a filter program is substituted using the
letter "N"
in nucleotide sequence (e.g., "NNNNNNNNNNNNN") and the letter "X" in protein
sequences (e.g., "XXXXXXXXX").
Filtering is only applied to the query sequence (or its translation products),
not to
database sequences. Default filtering is DUST for BLASTN, SEG for other
programs.
It is not unusual for nothing at all to be masked by SEG, XNU, or both, when
applied
to sequences in SWISS-PROT, so filtering should not be expected to always
yield an
effect. Furthermore, in some cases, sequences are masked in their entirety,
indicating
that the statistical significance of any matches reported against the
unfiltered query
sequence should be suspect.
NCBI-gi Causes NCBI gi identifiers to be shown in the output, in addition to
the
accession and/or locus name.
Most preferably, sequence comparisons are conducted using the simple BLAST
search
algorithm provided at http://www.ncbi.nlm.nih.govIBLAST.
The various classes of chaperone proteins are generally homologous in
structure and so
2S there are therefore conserved or substantially homologous amino acid
sequences
between the members of the class. For instance, GroEL is just an example of an
hsp60
chaperonin protein; other suitable proteins having an homologous apical domain
may be
followed.
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24
The list was compiled from the OWL database release 28.1. The sequences listed
below show clear homology to apical domain (residues 19I-375) in PDB structure
pdb 1 grl . ent.
OWL is a non redundant database merging SWISS-PROT, PIR (1-3)> GenBank
(translation) and NRL-3D.
190-374 CH60 ECOLI 60 KD CHAPERONIN (PROTEIN CPN60)(GROEL
PROTEIN)(AMS). - ESCHERICHIA 190-374 CH60 SALTI 60 KD
CHAPERONIN (PROTEIN CPN60)(GROEL PROTEIN). - SALMONELLA
TYPHI. 191-375 S56371 GroEL protein - Escherichia coli 190-
374 CH60 LEPIN 60 KD CHAPERONIN (PROTEIN CPN60)(GROEL
PROTEIN)(HEAT SHOCK 58 KD PRO 191-375 547530 groEL protein -
Porphyromonas gingivalis 190-374 LPNHTPBG LPNHTPBG
NID:g149691 - Legionella pneumophila (strain SVir)(library:
189-373 CH60 ACTAC 60 KD CHAPERONIN (PROTEIN CPN60)(GROEL
PROTEIN). - ACTINOBACILLUS ACT 191-375 JC4519 heat-shock
protein GroEL - Pasteurella multocida
191-375 CH60 BRUAB 60 KD CHAPERONIN (PROTEIN CPN60)(GROEL
PROTEIN). - BRUCELLA ABORTUS. 191-375 CH60 HAEIN 60 KD
CHAPERONIN (PROTEIN CPN60)(GROEL PROTEIN).- HAEMOPHILUS
INFLUE 190-373 CH60_CAUCR 60 KD CHAPERONIN (PROTEIN
CPN60)(GROEL PROTEIN).- CAULOBACTER CRESCE 190-374
CH60 AMOPS 60 KD CHAPERONIN (PROTEIN CPN60)(GROEL PROTEIN).
AMOEBA PROTEUS SYM 191-375 CH60 HAEDU 60 KD CHAPERONIN
(PROTEIN CPN60)(GROEL PROTEIN). - HAEMOPHILUS DUCREY 191-375
CH61 RHIME 60 KD CHAPERONIN A (PROTEIN CPN60 A)(GROEL
PROTEIN A). - RHIZOBIUM ME 190-374 CH60 LEGMI 60 KD
CHAPERONIN (PROTEIN CPN60)(GROEL PROTEIN)(58 KD COMMON
ANTIGEN 191-375 CH60 YEREN 60 KD CHAPERONIN (PROTEIN
CPN60)(GROEL PROTEIN)(HEAT SHOCK PROTEIN 6) 190-374 CH
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63 BRAJA 60 KD CHAPERONIN 3 (PROTEIN CPN60 3)(GROEL PROTEIN
3). - BRADYRHIZOBI 191-375 CH60_PORGI 60 KD CHAPERONIN
(PROTEIN CPN60)(GROEL PROTEIN). - PORPHYROMONAS GING 191-375
552901 heat shock protein 60K - Yersinia enterocolitica
5 191-375 526423 heat shock protein 60 - Yersinia
enterocolitica
191-375 RSU373691 RSU37369 NID: 81208541 - Rhodobacter
sphaeroides strain=HR. 190-374 CH62 BRAJA 60 KD CHAPERONIN
2 (PROTEIN CPN60 2) (GROEL PROTEIN 2) . - BRADYRHIZOBI 191-375
10 CH60 ACYPS 60 KD CHAPERONIN (PROTEIN CPN60)(GROEL
PROTEIN)(SYMBIONIN). - ACYRTH 191-375 CH63 RHIME 60 KD
CHAPERONIN C (PROTEIN CPN60 C) (GROEL PROTEIN C) . - RHIZOBIUM
ME 191-375 YEPHSPCRP1 YEPHSPCRP NID: 8466575 - Yersinia
enterocolitica DNA. 191-375 CH60 BORPE 60 KD CHAPERONIN
15 (PROTEIN CPN60)(GROEL PROTEIN). - BORDETELLA PERTUSS 189-373
BRUGRO1 BRUGRO NID: 8144106 - 8rucella aabortus (library:
lambda-2001) DNA.
191-375 CH60 PSEAE 60 KD CHAPERONIN (PROTEIN CPN60)(GROEL
PROTEIN). - PSEUDOMONAS AERUGI 190-374 CH60 BARBA 60 KD
20 CHAPERONIN (PROTEIN CPN60)(IMMUNOREACTIVE PROTEIN
BB65)(IMMUNO 191-375 BAOBB63A BAOBB63A NID: 8143845 -
Bartonella bacilliformis (library: ATCC 35685) 189-373
CH60 BACST 60 KD CHAPERONIN (PROTEIN CPN60)(GROEL PROTEIN).
- .BACILLUS STEAROTHE 188-372
25 190-373 CH60 BORBU 60 KD CHAPERONIN (PROTEIN CPN60)(GROEL
PROTEIN). - BORRELIA BUR.GDORFE 224-408 526583 chaperonin
hsp60 - maize 190-373 A49209 heat shock protein HSP60 - Lyme
disease spirochete 224-408 MZECPN60B MZECPN60B NID: 8309558
- Zea mays (strain B73?(library:Dashll of P.S 189-373
CH60 THEP3 60 KD CHAPERONIN (PROTEIN CPN60)(GROEL
PROTEIN)(HEAT _SHOCK 61 KD PRO 188-372 CH60 STAEP 60 KD
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26
CHAPERONIN (PROTEIN CPN50)(GROEL PROTEIN)(HEAT SHOCK PROTEIN
6 189-373 CH60 LACLA 60 KD CHAPERONIN (PROTEIN CPN60)(GROEL
PROTEIN). - LACTOCOCCUS LACTIS 188-374 CH61-STRAL 60 KD
CHAPERONIN 1 (PROTEIN CPN60 1)(GROEL PROTEIN 1)(HSP58). -
STRE 191-375 CH60_CHLPN 60 KD CHAPERONIN (PROTEIN
CPN60)(GROEL PROTEIN). - CHLAMYDIA PNEUMONI 224-408
MZECPN60A MZECPN60A NID: 8309556 - Zea mat's (strain
B73)(library:Dach 11 of P. 190-373 HECHSPAB1 HECHSPAB NID:
8712829 - Helicobacter pylori (individual-isolate 85P) D
221-405 CH60 ARATH MITOCHONDRIAL CHAPERONIN HSP60 PRECURSOR.
- AR.ABIDOPSIS THALIANA (MOUS 224-408 CH60 MAIZE
MITOCHONDRIAL CHAPERONIN HSP60 PRECURSOR. - ZEA MAYS
(MAIZE). 190-374 CH60-CHLTR 60 KD CHAPERONIN (PROTEIN
CPN60)(GROEL PROTEIN)(57 KD CHLAMYDIAL HYP 189-373
CH60 STAAU 60 KD CHAPERONIN (PROTEIN CPN60)(GROEL
PROTEIN)(HEAT SHOCK PROTEIN 6 189-373 CH60-CLOPE 60 KD
CHAPERONIN (PROTEIN CPN60)(GROEL PROTEIN). - CLOSTRIDIUM
PERFRI 212-397 HS60 YEAST HEAT SHOCK PROTEIN 60 PRECURSOR
(STIMULATOR FACTOR 1 66 KD COMPONENT) 217-403 CH60-PYRSA 60
KD CHAPERONIN (PROTEIN CPN60)(GROEL PROTEIN). - PYRENOMONAS
SALINA 191-377 CH60 EHRCH 60 KD CHAPERONIN (PROTEIN
CPN60)(GROEL PROTEIN).- EHRLICHIA CHAFFEEN 191-375 CHTGROE1
CHTGROE NID: 8144503 - C.trachomatis DNA. 188-372 CH60_THETH
60 KD CHAPERONIN(PROTEIN CPN60)(GROEL PROTEIN). - THERMUS
AQUATICUS 189-373 TAU294831 TAU29483 NID: 81122940 - Thermus
aquaticus. 190-378 CH60 RICTS 60 KD CHAPERONIN (PROTEIN
CPN60)(GROEL PROTEIN)(MAJOR ANTIGEN 58)(5 189-375 SYCCPNC
SYCCPNC NID: 81001102 - Synechocystis sp. (strain PCC6803,)
DNA.
190-373 CPU308211 CPU30821 NID: 81016083 - Cyanophora
paradoxa. 189-373 CH61 MYCLE 60 KD CHAPERONIN 1 (PROTEIN
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27
CPN60 1)(GROEL FROTEIN 1). - MYCOBACTERIU 239-423 PSU21139
PSU21139 NID: 8806807 - pea. 191-377 CH60_COWRU 60 KD
CHAPERONIN (PROTEIN CPN60)(GROEL PROTEIN). - COWDRIA
RUMINANTIU 245-429 RUBE BRANA RUBISCO SUBUNIT BINDING-
' S PROTEIN BETA SUBUNIT PRECURSOR (60 KD CHAPERON 144-328
SCCPN60 SCCPN60 NID: 81167857 - rye.
153-338 CH60 EHRRI 60 KD CHAPERONIN (PROTEIN CPN60)(GROEL
PROTEIN)(55 KD MAJOR ANTIGEN) 245-429 RUBB ARATH RUBISCO
SUBUNIT BINDING-PROTEIN BETA SUBUNIT PRECURSOR (60 KD
CHAPERON 235-419 ATU49357 ATU49357 NID: 81223909 - thale
cress strain=ecotype Wassilewskija. 195-379 RUB1 BRANA
RUBISCO SUBUNIT BINDING-PROTEIN ALPHA SUBUNIT (60 KD
CHAPERONIN ALPHA 189-374 CH62 SYNY3 60 KD CHAPERONIN 2
(PROTEIN CPN60 2)(GROEL HOMOLOG 2). - SYNECHOCYSTI 178-362
RUBA RICCO RUBISCO SUBUNIT BINDING-PROTEIN ALPHA SUBUNIT (60
KD CHAPERONIN ALPHA 190-375 CH60 ODOSI 60 KD CHAPERONIN
(PROTEIN CPN60)(GROEL PROTEIN). - ODONTELLA SINENSIS 236-420
PSU21105 PSU21105 NID: 81185389 - pea. 224-409 CH60 BRANA
MITOCHONDRIAL CHAPERONIN CH60 BACSU 60 KD CHAPERONIN
(PROTEIN CPN60)(GROEL PROTEIN). - BACILLUS SUBTILIS. 191-375
CH60 AGRTU 60 KD CHAPERONIN (PROTEIN CPN60)(GROEL PROTEIN).
- AGROBACTERIUM TUME 191-375 b36917 heat shock protein GroEL
- Agrobacterium tumefaciens
191-375 PAU17072 PAU17072 NID: 8576778 - Pseudomonas
aeruginosa. 191-375 CH60 RHILV 60 KD CHAPERONIN (PROTEIN
CPN60)(GROEL PROTEIN). - RHIZOBIUM LEGUMINO 187-373
CH61 STRCO 60 KD CHAPERONIN 1 (PROTEIN CPN60 1)(GROEL
PROTEIN 1)(HSP58).- STRE 191-375 CH60-COXBU 60 KD CHAPERONIN
(PROTEIN CPN60)(GROEL PROTEIN)(HEAT SHOCK PROTEIN B 191-375
CH62 RHIME 60 KD CHAPERONIN B (PROTEIN CPN60 B)(GROEL
PROTEIN B). - RHIZOBIUM ME 191-375 PSEGROESL1 PSEGROESL NID:
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28
g151241 - Pseudomonas aeruginosa (library: ATCC 27853) 189-
372 CH61 SYNY3 60 KD CHAPERONIN 1 (PROTEIN CPN60 1)(GROEL
HOMOLOG 1).-SYNECHOCYSTI 189-373 CH60-CLOTM 60 KD CHAPERONIN
(PROTEIN CPN60)(GROEL PROTEIN)(HSP-60). - CLOSTRIDI 191-373
CH60 PSEPU 60 KD CHAPERONIN (PROTEIN CPN60)(GROEL PROTEIN).-
PSEUDOMONAS PUTIDA 190-373 CH60-SYNP7 60 KD CHAPERONIN
(PROTEIN CPN60)(GROEL PROTEIN).- SYNECHOCOCCUS SP. 190-374
CH60 GALSU 60 KD CHAPERONIN (PROTEIN CPN60)(GROEL PROTEIN).-
GALDIERIA SULPHURA 190-374 CH60_ZYMMO 60 KD CHAPERONIN
(PROTEIN CPN60)(GROEL PROTEIN). - ZYMOMONAS MOBILIS. 191-375
JC2564 heat shock protein groEL - Zymomonas mobilis
191-375 CH60 CHRVI 60 KD CHAPERONIN (PROTEIN CPN60)(GROEL
PROTEIN). - CHROMATIUM VINOSUM 189-373 CH60 MYCTU 60 KD
CHAPERONIN (PROTEIN CPN60}(GROEL PROTEIN)(65 KD
ANTIGEN)(HEAT 191-375 CH60 NEIME 60 KD CHAPERONIN (PROTEIN
CPN60)(GROEL PROTEIN)(63 KD STRESS PROTEIN 189-373
CH60 TREPA 60 KD CHAPERONIN (PROTEIN CPN60)(GROEL
PROTEIN)(TPN60)(TP4 ANTIGEN) 190-374 CH60 HELPY 60 KD
CHAPERONIN (PROTEIN CPN60)(GROEL PROTEIN)(HEAT SHOCK PROTEIN
6 191-37S CH60 NEIGO 60 KD CHAPERONIN (PROTEIN CPN60)(GROEL
PROTEIN)(63 KD STRESS PROTEIN 222-406 CH61-CUCMA
MITOCHONDRIAL CHAPERONIN HSP60-1 PRECURSOR. - CUCURBITA
MAXIMA (PUMPKI 189-373 CH60 MYCPA 60 KD CHAPERONIN (PROTEIN
CPN60)(GROEL PROTEIN)(65 KD ANTIGEN)(HEAT 230-414 MPU15989
MPU15989 NID:g559802 - Mycobacterium paratuberculosis. 224-
408 526582 chaperonin hsp60 - maize 191-375 540247 heat-
shock protein - Neisseria gonorrhoeae 189-373 CH60,CLOAB 60
KD CHAPERONIN (PROTEIN CPN60)(GROEL PROTEIN). - CLOSTRIDIUM
ACETOB 191-375 CH60 NEIFL 60 KD CHAPERONIN (PROTEIN
CPN60)(GROEL PROTEIN)(63 KD STRESS PROTEIN 190-373
CH60 LEGPN 60 KD CHAPERONIN (PROTEIN CPN60)(GROEL
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29
PROTEIN)(58 KD COMMON ANTIGEN 222-406 CH62-CUCMA
MITOCHONDRIAL CHAPERONIN HSP60-2 PRECURSOR. - CUCURBITA
. MAXIMA (PUMPKI 191-375 CHTGROESL1 CHTGROESL NID: 8402332
Chlamydia trachomatis DNA. 64-248 540172 S40172 NID:
8251679 - Chlamydia psittaci pigeon strain P-1041. 189-373
SYOGROEL2 SYOGROEL2 NID:g562270 - Synechococcus vulcanus
DNA. 191-375 CH60 CHLPS 60 KD CHAPERONIN (PROTEIN
CPN60)(GROEL PROTEIN)(57 KD CHLAMYDIAL HYP 188-372
CH62 STRAL 60 KD CHAPERONIN 2 (PROTEIN CPN60 2)(GROEL
PROTEIN 2)(HSP56). - STRE 189-373 CH62 MYCLE 60 KD
CHAPERONIN 2 (PROTEIN CPN60 2)(GROEL PROTEIN 2)(65 KD
ANTIGEN) 236-420 MSGANTM MSGANTM NID: 8149923 - M.leprae
DNA, clone Y3178.
CPN60 PRECURSOR. - BRASSICA NAPUS (RAPE). 105-289 PMSARG2 .
PMSARG2 NID: 8607157 - Prochlorococcus marinus.
234-417 RUB2 BRANA RUBISCO SUBUNIT BINDING-PROTEIN ALPHA
SUBUNIT PRECURSOR (60 KD CHAPERO 75-259 CRECPN1A CRECPN1A
NID: 8603910 - Chlamydomonas reinhardtii cDNA to mRNA. 215-
400 P60 CRIGR MITOCHONIDRIAL MATRIX PROTEIN P1 PRECURSOR
(P60 LYMPHOCYTE PROTEIN)(CH224-408 CRECPN1B CRECPN1B NID:
8603912 - Chlamydomonas reinhardtii cDNA to mRNA. 191-375
RUBA WHEAT RUBISCO SUBUNIT BINDING-PROTEIN ALPHA SUBUNIT
PRECURSOR (60 KD CHAPERO 189-373 B47292 heat shock protein
groEL - Mycobacterium tuberculosis
206-391 CELHSP60CP CELHSP60CP NID: 8533166 - Caenorhabditis
elegans (strain CB1392) cDNA 215-400 P60 HUMAN MITOCHONDRIAL
MATRIX PROTEIN P1 PRECURSOR (P60 LYMPHOCYTE PROTEIN)(CH 215-
400 P60 MOUSE MITOCHONDRIAL MATRIX PROTEIN P1 PRECURSOR (P60
LYMPHOCYTE PROTEIN)(CH 215-400 P60 RAT MITOCHONDRIAL MATRIX
PROTEIN P1 PRECURSOR (P60 LYMPHOCYTE PROTEIN)(CH 215-400
A41931 chaperonin hsp60 - mouse
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197-382 MMHSP60A MMHSP60A NID:g51451 - house mouse. 218-402
CH63 HELVI 63 KD CHAPERONIN PRECURSOR (P63). - HELIOTHIS
VIRESCENS (NOCTUID MOTH) 205-390 EGHSP60GN EGHSP60GN NID:
81217625 - Euglena gracilis. 222-407 HS60_SCHPO PROBABLE
5 HEAT SHOCK PROTEIN 60 PRECURSOR. - SCHIZOSACCHAROMYCES POMBE
198-385 S61295 heat shock protein 60 - Trypanosoma cruzi
198-385 TRBMTHSP TRBMTHSP NID: 8903883 - Mitochondrion
Trypanosoma brucei (strain EATRO 8-69 ECOGROELA ECOGROELA
NID: 8146268 - E.coli DNA, clone E. 142-325 ENHCPN60P
10 ENHCPN60P NID: 8675513 - Entamoeba histolytica (strain HM-
1:IMSS) DNA. 257-433 CH60_PLAFG MITOCHONDRIAL CHAPERONIN
CPN60 PRECURSOR. - PLASMODIUM FALCIPARUM (ISO 1-90 CRECPN1C
CRECPN1C NID: 8603914 - Chlamydomonas reinhardtii cDNA to
mRNA.
15 5-65 ATTS0779 ATTS0779 NID: 817503 - thale cress.
189-373 CH60 MYCGE 60 KD CHAPERONIN (PROTEIN CPN60)(GROEL
PROTEIN). - MYCOPLASMA GENITAL 228-411 HTOHSP60X HTOHSP60X
NID: 8553068 - Histoplasma capsulatum (strain G217B) DNA.
190-297 CH60 SYNP6 60 KD CHAPERONIN (PROTEIN CPN60)(GROEL
20 PROTEIN)(FRAGMENT). - SYNECHO 169-245 RUBA ARATH RUBISCO
SUBUNIT BINDING-PROTEIN ALPHA SUBUNIT (60 KD CHAPERONIN
ALPHA.
Such analyses may be repeated using other databases, or more recent updates of
the
25 OWL database, and for other chaperone families, such as the HSP 70, HSP 90
or GRP
families.
In a further aspect, the invention provides a method for providing a chaperone
activity
in vivo, comprising administering to a cell a fragment of a molecular
chaperone which
30 has in vivo activity.
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31
Fragments of molecular chaperones which possess in vivo activity may be
identified by
the method of the first aspect of the invention. Preferred fragments are GroEL
fragments 191-345 and I93-335.
Fragments according to the invention are capable of complementing deficiencies
in the
endogenous molecular chaperones of cells and are therefore particularly suited
for
rectifying defects caused by chaperone deficiencies. Preferably, fragments of
molecular
chaperones having in vivo activity may be administered to patients suffering
from
disorders or diseases associated with anomalies in protein folding or other
chaperone
functions .
The active ingredients of a pharmaceutical composition comprising the
chaperone
fragment are contemplated to exhibit excellent therapeutic activity, for
example, in the
alleviation of Alzheimer's disease when administered in amount which depends
on the
particular case. Dosage regima may be adjusted to provide the optimum
therapeutic
response. For example, several divided doses may be administered daily or the
dose
may be proportionally reduced as indicated by the exigencies of the
therapeutic
situation.
The active compound may be administered in a convenient manner such as by the
oral,
intravenous (where water soluble), intramuscular, subcutaneous, intranasal,
intradermal
or suppository routes or implanting (e.g. using slow release molecules).
Depending on
the route of administration, the active ingredient may be required to be
coated in a
material to protect said ingredients from the action of enzymes, acids and
other natural
conditions which may inactivate said ingredient.
In order to administer the polypeptide by other than parenteral
administration, .it will be
coated by, or administered with, a material to prevent its inactivation. For
example,
the polypeptide may be administered in an adjuvant, co-administered with
enzyme
inhibitors or in liposomes. Adjuvant is used in its broadest sense and
includes any
immune stimulating compound such as interferon. Adjuvants contemplated herein
CA 02290038 1999-11-10
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include resorcinols, non-ionic surfactants such as polyoxyethylene oleyl ether
and n-
hexadecyl polyethylene ether. Enzyme inhibitors include pancreatic trypsin.
Liposomes include water-in-oil-in-water CGF emulsions as well as conventional
liposomes.
The active compound may also be administered parenterally or
intraperitoneally.
Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and
mixtures
thereof and in oils. Under ordinary conditions of storage and use, these
preparations
contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous
solutions
(where water soluble) or dispersions and sterile powders for the
extemporaneous
preparation of sterile injectable solutions or dispersion. In all cases the
form must be
sterile and must be fluid to the extent that easy syringability exists. It
must be stable
under the conditions of manufacture and storage and must be preserved against
the
contaminating action of microorganisms such as bacteria and fungi. The carrier
can be
a solvent or dispersion medium containing, for example, water, ethanol, polyol
(for
example, glycerol, propylene glycol, and liquid polyetheyiene gloycol, and the
like),
suitable mixtures thereof, and vegetable oils. The proper fluidity can be
maintained,
for example, by the use of a coating such as lecithin, by the maintenance of
the
required particle size in the case of dispersion and by the use of
superfactants.
The prevention of the action of microorganisms can be brought about by various
antibacterial and antifungal agents, for example, parabens, chlorobutanol,
phenol,
sorbic acid, thirmerosal, and the like. In many cases, it will be preferable
to include
isotonic agents, for example, sugars or sodium chloride. Prolonged absorption
or the
injectable compositions can be brought about by the use in the compositions of
agents
delaying absorption, for example, aluminium monostearate and gelatin.
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33
Sterile injectable solutions are prepared by incorporating the active compound
in the
required amount in the appropriate solvent with various of the other
ingredients
enumerated above, as required, followed by filtered sterilisation. Generally,
dispersions are prepared by incorporating the sterilised active ingredient
into a sterile
vehicle which contains the basic dispersion medium and the required other
ingredients
from those enumerated above. In the case of sterile powders for the
preparation of
sterile injectable solutions, the preferred methods of preparation are vacuum
drying and
the freeze-drying technique which yield a powder of the active ingredient plus
any
additional desired ingredient from previously sterile-filtered solution
thereof.
When the chaperone fragment is suitably protected as described above, it may
be orally
administered, for example, with an inert diluent or with an assimilable edible
carrier, or
it may be enclosed in hard or soft shell gelatin capsules, or it may be
compressed into
tablets, or it may be incorporated directly with the food of the diet. For
oral
therapeutic administration, the active compound may be incorporated with
excipients
and used in the form of ingestible tablets, buccal tablets, troches, capsules,
elixirs,
suspensions, syrups, wafers, and the like. The amount of active compound in
such
therapeutically useful compositions in such that a suitable dosage will be
obtained.
The tablets, troches, pills, capsules and the like may also contain the
following: a
binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such
as
dicalcium phosphate; a disintegrating agent such as corn starch, potato
starch, alginic
acid and the like; a lubricant such as magnesium stearate; and a sweetening
agent such
as sucrose, lactose or saccharin may be added or a flavouring agent such as
peppermint,
oil of wintergreen, or cherry flavouring. When the dosage unit form is a
capsule, it
may contain, in addition to materials of the above type, a liquid carrier.
Various other materials may be present as coatings or to otherwise modify the
physical
form of the dosage unit. For instance, tablets, pills, or capsules may be
coated with
shellac, sugar or both. A syrup or elixir may contain the active compound,
sucrose as
a sweetening agent, methyl and propylparabens as preservatives, a dye and
flavouring
CA 02290038 1999-11-10
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34
such as cherry or orange flavour. Of course, any material used in preparing
any dosage
unit form should be pharmaceutically pure and substantially non-toxic in the
amounts
employed. In addition, the active compound may be incorporated into sustained-
release
preparations and formulations.
As used herein "pharmaceutically acceptable carrier andlor diluent" includes
any and all
solvents, dispersion media, coatings, antibacterial and antifungal agents,
isotonic and
absorption delaying agents and the like. The use of such media and agents for
pharmaceutical active substances is well known in the art. Except insofar as
any
conventional media or agent is incompatible with the active ingredient, use
thereof in
the therapeutic compositions is contemplated. Supplementary active ingredients
can
also be incorporated into the compositions.
It is especially advantageous to formulate parenteral compositions in dosage
unit form
for ease of administration and uniformity of dosage. Dosage unit form as used
herein
refers to physically discrete units suited as unitary dosages for the
mammalian subjects
to be treated; each unit containing a predetermined quantity of active
material calculated
to produce the desired therapeutic effect in association with the required
pharmaceutical
carrier. The specification for the novel dosage unit forms of the invention
are dictated
by and directly dependent on (a) the unique characteristics of the active
material and the
particular therapeutic effect to be achieved, and (b} the limitations inherent
in the art of
compounding such as active material for the treatment of disease in living
subjects
having a diseased condition in which bodily health is impaired.
The principal active ingredient is compounded for convenient and effective
administration in effective amounts with a suitable pharmaceutically
acceptable carrier
in dosage unit form. In the case of compositions containing supplementary
active
ingredients, the dosages are determined by reference to the usual dose and
manner of
administration of the said ingredients.
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WO 99/02989 PCT/GB98/02042
In a preferred aspect there is provided polypeptide of the invention as
hereinbefore
defined for use in the treatment of disease. Consequently there is provided
the use of a
chaperone fragment of the invention for the manufacture of a medicament for
the
treatment of disease associated with aberrant protein/polypeptide structure.
The
S aberrant nature of the proteinlpolypeptide may be due to misfolding or
unfolding which
in turn may be due to an anomalous e.g. mutated amino acid sequence. The
protein/polypeptide may be destabilised or deposited as plaques e.g. as in
Alzheimer's
disease. The disease might be caused by a prion. A polypeptide-based
medicament of
the invention would act to renature or resolubilise aberrant, defective or
deposited
10 proteins.
In a further aspect, there is provided a nucleic acid molecule encoding a
chaperone
fragment in accordance with other aspects of the invention for use in the
treatment of
disease. Consequently, there is provided the use of a nucleic acid molecule of
the
15 invention for the manufacture of a medicament for the treatment of disease
associated
with proteinlpolypeptide structure. Genetic therapy in vivo is therefore
provided for by
way of introduction and expression of DNA encoding the chaperone fragment in
ceilsltissues of an individual to provide chaperonin activity in those
cells/tissues.
20 The invention is further described, for the purposes of illustration only,
in the following
examples.
Materials and Methods
25 Bacterial and bacteriophage strains. The E. coli strains used in this study
are:
C41(DE3), a mutant of BL21(DE3) capable of expressing toxic genes (Miroux, B.
&
Walker, J. E. (1996) J. Mol. Biol. 260, 289-298) ; SV2 (B178groEL44) and SV6
(B178groEL673): isogenic strains carrying temperature-sensitive alleles of
groEL;
SV1(=B178) (24), AI90 (~groEL::kanR) [pBAD-EL] (Ivic, A., Olden, D.,
Wellington,
30 E. J. & Lund, P. A. (1997) Gene 194, 1-8) , and TG1 (Gibson, T. J. (1984)
Ph.D.
thesis, University of Cambridge). ~, b2cI (Zeilstra-Ryalls, J., Fayet, O.,
Baird, L. &
CA 02290038 1999-11-10
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36
Georgopoulos; C. (1993) J. Bacteriol. 175, 1134-1143) is used, according to
standard
methods (Arber, W., Enquist, L., Hohn, B., Murray, N. E. & Murray, K. (1983)
in
Lambda ll, eds. Hendrix, R., Roberts, J., Stahl, F. & Weisberg, R. (Cold
Spring
Harbor Laboratory, N.Y.), pp. 433-466) . Plaque formation is assayed at
30°C.
Plasmid construction. Standard molecular biology procedures are used
(Sambrook, J.,
Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual
(Cold
Spring Harbor Laboratory, N.Y.)) . The principal steps of plasmid construction
are
summarised in Fig. 1.
Genomic DNA is extracted from E. coli TG1 (Gibson, T. 3. (1984) Ph.D. thesis,
University of Cambridge) using a commercial kit (Qiagen). PCR using two
oligonucleotides 5' - ATT C',AT ATG AAT ATT CGT CCA TTG CAT GAT CG - 3'
(SEQ. ID. No. 1) and 5' - AA C'.GG CCG TTA ATT AAG GTG CAC CGA AAG ATT
TAT CCA GAA CTA CG - 3' (SEQ. ID. No. 2) produce a 494 by DNA carrying the
complete 294 by of groES gene and unique sites for Nde I and Eag I
(underlined). This
fragment also contains the 44 by intergenic ES-EL region and the first 123 by
of
groEL, including the unique ApaL I site (bold characters) of the groE operon
(Hemmingsen, S. M., Woolford, C., van der Vies, S., Tilly, K., Dennis, D. T.,
Georgopoulos, C. P., Hendrix, R. W. & Ellis, R. J. (1988) Nature 333, 330-
334).
The GroEL gene (SEQ. ID. No. 10) is PCR amplified using two oligonucleotides
5' - T
AGC TGC CAT ATG GCA GCT AAA GAC GTA AAA TTC GG - 3' (SEQ. ID. No.
3) and 5' - ATG TAA ('-~G CCG TTA CAT CAT GCC GCC CAT GCC ACC - 3'
(SEQ. ID. No. 4) producing a 1,659 by DNA with unique sites for Nde I and Eag
I
(underlined). These fragments are cloned into pRSETA-Eag I allowing expression
of
GroE proteins under control of a T7 promoter. The unique EagI recognition site
(underlined) is created by replacing the EcoR I-Hind III fragment of pRSETA
(InVitrogen) using a synthetic DNA cassette consisting of two oligos 5' - AAT
TCA A
GGG CCG TTA - 3' (SEQ. ID. No. S) and 5' - AGC TTA AC'.(: GCC GTT G- 3' (SEQ.
ID. No. 6). The pRSETA-Eag I (GroESL) vector is generated by subcloning the
Nde
IlApaL I fragment from pRSETA-Eag I (GroES) into pRSETA-Eag I (GroEL) OlVde I-
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37
ApaL I. Various N- and C-terminally truncated fragments of the apical domain
of
GroEL are cloned by PCR into BamH I and Eag I sites of pRSETA-Eag I vector
encoding an N-terminal histidine-tail (17 amino acids; "sht"),
MRGSHHHHHHGLVPRGS (SEQ. ID. No. 7), which contains an engineered thrombin
cleavage site (Zahn, R., Buckle, A. M., Perret, S., Johnson, C. M. J.,
Corrales, F. J.,
Golbik, R. & Fersht, A. R. (1996) Proc. Natl. Acad. Sci. USA 93, 15024-15029)
.
The mutation Y203E is introduced into GroEL by PCR, as described (Hemsley, A.,
Arnheim, N., Toney, M. D., Cortopassi, G. & Galas, D. J. (1989) Nucleic Acids
Res.
17, 6545-6551) , using oligonucleotides 5' - ATT CAT CAA CAA GCC - 3' (SEQ.
ID.
No. 8) and 5' - TCA GGA GAC AGG TAG CC - 3' (SEQ. ID. No. 9), creating a
unique EcoR I site (bold characters).
A modified pACYC184 vector (New England Biolabs) is constructed. The different
pRSETA-Eag I based vectors are digested by Xba I, the recessed 3' ends filled
in with
Klenow enzyme and then, digested by Eag I. The Xba I blunt-ended-Eag I
fragments,
containing the ribosome binding site of pRSETA, are ligated into pACYC184 EcoR
V l
Eag I digested and alkaline phosphatase treated plasmid. pJC vector is
generated by
replacing the Xmn II Ase I fragment of pACYCI84 by the Ase I-BsaB I of pBR322
prepared in the dam- dcm- JM110 E. toll strain (Yanisch-Perron, C., Viera, J.
&
Messing, J. (1985) Gene 33, 103-119) . The Xba I l Hind III fragments from
pRSETA-Eag I based vectors are cloned into pBAD30 Xba I I Hind III digested
and
alkaline phosphatase treated plasmid. Alternatively, the different groE genes
are
subcloned into the unique Nde I and Eag I unique sites of pACYC 184 or pJC and
pBAD30 (Guzman, L.-M., Belin, D., Carson, M. J. & Beckwith, 3. (1995) J.
Bacteriol. 177, 4121-4130) vectors. Plasmids containing no groE inserts are
generated
by ligated filled-in recessed 3' ends of pACYC 184 or pJC and pBAD30 ~lVde I-
Eag I
vectors .
A colony-based PCR procedure (Gussow, D. & Clackson, T. (1989) Nucleic Acids
Res.
I7: 4000) is performed to identify the positive clones using T7 promoter and
3' reverse
cloning oligonucleotides. PCR cycle sequencing using fluorescent dideoxy chain
CA 02290038 1999-11-10
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38
terminators (Applied Biosystems) are performed. Sequencing reactions are
analysed on
an Applied Biosystems 373A Automated DNA. All PCR amplified DNA fragments are
sequenced after cloning. The groEL gene differs from the sequence in the
database
(Hemmingsen, S. M., Woolford, C., van der Vies, S., Tilly, K., Dennis, D. T.,
Georgopoulos, C. P., Hendrix, R. W. & Ellis, R. J. (1988} Nature 333, 330-334)
by
the substitutions A262L and I267M as described (Zeilstra-Ryalls, J., Fayet,
O., Baird,
L. & Georgopoulos, C. (1993) J. Bacteriol. 175, l I34-1143) .
Protein production and characterisation. GroE proteins, - 57 .5 kDa GroEL and
--10 kDa GroES, are expressed by inducing the T7 promoter of pRSETA-Eag I
based
vectors with isopropyl-~i-D-thiogalactoside (IPTG) in E. coli C41(DE3)
(Miroux, B. &
Walker, J. E. (1996) J. Mol. Biol. 260, 289-298) . Purification is performed
as
previously described (Corrales, F. J. & Fersht, A. R. (1996) Folding & Design
1, 265-
273) . The over-expression and purification of minichaperones in E. coli
C41(DE3)
cells is carried out essentially as previously described (Zahn, R., Buckle, A.
M.,
Perret, S., Johnson, C. M. J., Corrales, F. J., Golbik, R. & Fersht, A. R.
(1996)
Proc. Natl. Acad. Sci. USA 93, 15024-15029). Proteins are analysed by
electrospray
mass spectrometry. Protein concentration is determined by absorbance at 276 nm
using
the method of Gill & von Hippel (Gill, S. C. & von Hippel, P. H. (/989)
Analyt.
Biochem. 182, 319-326) and confirmed by quantitative amino acid analysis.
Constitutive expression under the control of the tetracycline-resistance gene
promoter
operator is obtained either using the low copy-number pACYCl84 or the high
copy-
number pJC vectors. pBAD30 vector allows inducible expression with 0.2-0.5 %
arabinose controlled by the PBS promoter and its regulatory gene, araC
(Guzman,
L.-M., Belin, D., Carson, M. J. & Beckwith, J. (1995) J. Bacteriol. 177, 4121-
4130).
The level of expression of GroEL minichaperones is analysed by 15 % sodium
dodecyl
sulphate polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing
conditions followed by Western blotting. After separation of proteins, GroEL
molecules are detected with rabbit anti-GroEL antibodies (Sigma) followed by
anti-
rabbit immunoglobulins horseradish peroxidase conjugate antibodies (Sigma).
CA 02290038 1999-11-10
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39
Bromochloroindolyl phosphate {BCIP) I vitro blue tetrazolium (NBT) chromogens
(Sigma) are used as substrate.
In vitro refolding experiments. Refolding assays of rhodanese (Horowitz, P. M.
(1995) in Chaperonin-assisted protein folding of the enryme rhodanese by
GroELlGroES, eds. Shirley, B. A. (Humans Press), Vol. 40, pp. 361-368) and
cyclophilin A (Zahn, R., Buckle, A. M., Perret, S., Johnson, C. M. J.,
Corrales, F.
J., Golbik, R. & Fersht, A. R. (1996) Proc. Natl. Acad. Sci. USA 93, 15024-
15029)
are carried out as described.
In vivo complementation experiments. Complementation experiments at
43°C are
performed by transforming the thermosensitive (ts) E. coli strains SV2 or SV6
(Zeilstra-Ryalls, J., Fayet, O., Baird, L. & Georgopoulos, C. (1993) J.
Bacteriol. 175,
1134-1143) with the pJC or pBAD30 series of expression vectors. 3 ml cultures
of
appropriately transformed cells are grown o/n at 30°C in LB. Cells
(A600 = 0.1) are
serially diluted 10-fold in sterile 0.85 % (wlv) NaCI. 5 ~,L from each 10-fold
dilution is
spotted onto each of two LB plates. One plate is incubated oln at 37°C
and the other
overnight at 43°C. The number of viable cells/ml of culture is deduced
from the
number of colonies in the lowest dilution to give single colonies.
P1 transduction (35) , using strain AI90 (OgroEL::kanR) [pBAD-EL] as donor
(Ivic,
A., Olden, D., Wallington, E. J. & Lund, P. A. (1997) Gene 194, 1-8), is used
to
delete the groEL gene of TG1 cells transfected by the different pACYC184 or
pJC or
pBAD30 (sht-GroEL minichaperones) vectors. Transductants are selected on LB
plates
containing 10 ~glml of kanamycin at 37°C. Approximately 25 colonies are
transferred
onto plates containing kanamycin at 50 ~,g/mi. After incubation for 24 h at
37°C,
colonies that grew are screened by PCR as described (Ivic, A., Olden, D.,
Wallington,
E. J. & Lund, P. A. (1997) Gene 194, 1-8). AI90 (~groEL::kanR) [pBAD-EL] cells
are transformed with the pJC(sht-GroEL minichaperones) vectors. Transformants
are
selected at 37°C on LB supplemented with 50 ~,g/ml of kanamycin, 120
~g/ml of
ampicillin, 25 ~glml of chloramphenicol and 0.2% L(+)arabinose. Depletion of
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GroEL protein is analysed at 37°C by plating the same quantity of AI90
[pBAD-EL +
pJC (sht-GroEL minichaperones)] cells on LB plates containing 1 % D(+)alucose
or
various amount of arabinose.
5 Each experiment is performed in triplicate. Plasmids carrying no groE genes
or
encoding the GroE proteins are used as negative or positive controls,
respectively.
Effect on Lorist6 replication of over-expressing GroE proteins or the
minichaperones. TG1 (Gibson, T. J. (1984) Ph.D. thesis, University of
Cambridge)
10 cells carrying the bacteriophage ~, origin vector, Lorist6 which encodes
kanamycin
resistance (Gibson, T. J., Rosenthal, A. & Waterston, R. H. (1987) Gene 53,
283-286)
are transformed with the pBAD30 series of plasmids. Transformants are selected
at
37°C on LB supplemented with 50 ~cglml of kanamycin, I20 ~.glml of
ampicillin and
1.0% D(+)glucose. 10-fold serial dilutions of overnight cultures are plated
onto
15 kanamycin + ampicillin LB with or without 0.2% L(+)arabinose. The effect of
over-
expressing GroE proteins or minichaperones on Lorist6 replication is
determined by
comparing the number of colonies forming units (cfu} per ml resistant to
kanamycin on
plates containing arabinose relative to the number formed on plates lacking
arabinose at
37°C.
Example 1
Preparation of chaperone fragments and analysis of in vitro folding activity
Identification of minimal GroEL chaperone fragment. The crystal structure of
the
active minichaperone GroEL(191-345), solved at 2.5 A, displays a well-ordered
domain
with the same fold as in intact GroEL chaperonin (Zahn, R., Buckle, A. M.,
Perret, S.,
Johnson, C. M. J., Corrales, F. J., Golbik, R. & Fersht, A. R. (/996) Proc.
Natl.
Acad. Sci. USA 93, 15024-15029) (Fig. 2). No electron density is observed for
the C-
terminal residues 337-345, corresponding to the first half of a-helix H I i .
Residue
GIy335 is at the end of a [3-strand. Further truncation before residue 329
leads to
inclusion-body formation indicating considerable destabilisation of the over-
expressed
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41
GroEL fragment. Residue G1u191 protrudes from the protein surface, making no
interactions. G1y192 NH C-caps a-helix H12 which is absent in the functional
monomeric minichaperone GroE(191-345). The N-terminal ~-strand starts at
Met193
in the hydrophobic core. The integrity of the hydrophobic core is considered
to
stabilise the polypeptide-binding site composed by a-helices H8 and H9 and the
surrounding loops (Buckle, A. M., Zahn, R. & Fersht, A. R. (1997) Proc. Natl.
Acad.
Sci. USA 94, 3571-3575; Fenton, W. A., Kashi, Y., Furtak, K. & Norwich, A. L.
(I994) Nature 371, 614-619) . On structural grounds, it is predicted that
GroEL(193-
335) should be the minimal active chaperone unit (Fig. 2).
Various N- and C-terminally truncated fragments of the apical domain of GroEL
are
amplified and cloned downstream of the T7 promoter of pRSETAsht-Eag I vector.
The
minichaperones sht-GroEL(i91-345) and sht-GroEL(191-376) have previously been
expressed with a short histidine tail ("sht") for ease of purification (Zahn,
R., Buckle,
A. M., Perret, S., Johnson, C. M. J., Corrales, F. J., Golbik, R. & Fersht, A.
R.
(1996) Proc. Natl. Acad. Sci. USA 93, 15024-15029). After sonication, the
soluble
fractions of IPTG-induced transfected C41(DE3) cells are analysed by SDS-PAGE.
sht-GroEL(193-335) is over-expressed in C41(DE3) cells (Miroux, B. & Walker,
J. E.
(1996) J. Mol. Biol. 260, 289-298) to give -150 mg purified protein per L of
culture.
Further truncations leads to severe aggregation in C41(DE3) cells. Fragments
(all with
sht) 193-335, 193-336, 193-337, I93-345, 191-335, 191-336, 191-337 and 191-345
are
all expressed at 100-150 mglL culture after purification and are highly
soluble. 193-
330, 195-330 and 195-335 are all poorly expressed and have low solubility. sht-
GroEL(193-335) purified by gel-filtration is monomeric at ~M-mM
concentrations, as
determined by light scattering and NMR experiments.
The minichaperone GroEL(193-335), lacking the N-terminal histidine-tail, is
generated
by thrombin cleavage of purified sht-GroEL(193-335). The circular dichroism
spectra
of GroEL(193-335) with or without the short histidine tail indicated
significant a-
helical structure. sht-GroEL(193-335) has been found by differential scanning
calorimetry experiments to be the most thermostable of the minichaperones with
a Tm
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at about 70°C. The calorimetric data are also consistent with the
unfolding of the
minichaperone sht-GroEL(193-335) as a monomer.
vitro activity of minichaperone GroEL(193-335). In vitro, rhodanese refolds in
high
yield only in the presence of GroEL, ATP, and the co-chaperonin GroES (Zahn,
R.,
Perrett, S., Stenberg, G. & Fersht, A. R. (1996) Science 27I, 642-645).
Minichaperone sht-GroEL(193-335) is as active in vitro as sht-GroEL(191-345)
and
more active than sht-GroEL(191-376) in chaperoning the folding of rhodanese
(Fig. 3).
However, all three chaperone fragments retain in vitro activity.
The presence of the N-terminal histidine-tail does not abolish binding
activity.
Residue Y203 is in the polypeptide-binding site of GroEL(19I-376)
minichaperone
(Fig. 2) (Buckle, A. M., Zahn, R. & Fersht, A. R. (1997) Proc. Natl. Acad.
Sci. USA
94, 3571-3575) . The mutation Y203E prevents the binding of intact GroEL to
the
human ornithine transcarbamylase (Fenton, W. A., Kashi, Y., Furtak, K. &
Horwich,
A. L. (1994) Nature 371, 614-619) . The mutation Y203E similarly abolishes the
chaperone activity of sht-GroEL(193-335) (Fig. 3). This suggests that the
recognition
of denatured polypeptide substrates by GroEL and minichaperones uses the same
residues.
Example 2
In vivo complementation by chaperone fragments of is chaperone mutants
To determine whether GroEL minichaperones can supplement defective GroEL in
general cell growth, complementation of two thermosensitive (ts) groEL mutants
of E.
coli at 43°C is examined (Fig. 4}. E. coli SV2 has the mutation G1u191--
~Gly in
GroEL corresponding to groEL44 allele , while SV6 carries the EL673 allele,
which
has two mutations, G1y173~Asp and G1y337~Asp(Zeilstra-Ryails, J., Fayet, O.,
Baird, L. & Georgopoulos, C. (1993) J. Bacteriol. 175, 1134-1143). The
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43
thermosensitivity at 43°C of the SV2 and SVb strains is suppressed by
transformation of
pJCGroESL containing the groE operon or by GroEL alone (Fig. 4).
Only the minichaperone sht-GroEL(193-335) complements the defect in SV2. About
' 5 4S % of the cells, transformed by the vector encoding the minichaperone
sht-
GroEL(193-335) which grow at the permissive temperature of 37°C, also
grow at 43°C
(Fig. 4). In contrast, the defective groEL in SV6 is complemented by
expression of the
minichaperone sht-GroEL(191-345), and less well by sht-GroEL(193-335) (see Fig
4).
About 65% of the cells SV6 transformed with pJCsht-GroEL(191-345), which grow
at
37°C, also grow at 43°C (Fig. 4). Colony-forming units are not
observed for either
strain at 43°C with vectors either lacking inserts or containing the
mutation, Y203E, in
the minichaperone gene. Thus, the mutation Y203E, which prevents the growth of
LG6 E. coli strain (Fenton, W. A., Kashi, Y., Furtak, K. & Horwich, A. L.
(1994)
Nature 371, 614-619), also inactivates the minichaperones in vivo. Similar
results are
obtained using the pBAD30(sht-GroELminichaperone) expression system.
Example 3
In vivo complementation at 37°C
The effects of minichaperones on the growth at 37°C of a strain of E.
coli in which the
chromosomal groEL gene has been deleted are analysed in two ways. First, the
groEL
gene of TG1 which had been transformed with the different pJC or pBAD30 (sht-
GroEL minichaperone) vectors is deleted. This is done using P1 transduction
from the
strain AI90 where the chromosomal groEL gene has been precisely replaced by a
kanamycin resistance (kanR) cassette (Ivic, A., Olden, D., Wallington, E. J. &
Lund,
P. A. (1997) Gene 194, 1-8) . However, no kanR transductants can be obtained
where
the groEL gene had been deleted, unless intact GroEL is expressed from the
complementing plasmid. This is consistent with the known essential role of
GroEL
(Fayet, O., Ziegelhoffer, T. & Georgopoulos, C. (1989) J. Bacteriol. 171, 1379-
1385).
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44
In a second series of experiments, the complementation of AI90 (~groEL::kanR)
(pBAD-EL] E. toll strain (Ivic, A., Olden, D., Wallington, E. J. & Lund, P. A.
(1997) Gene 194, 1-8) is analysed. In this strain, the chromosomal groEL gene
has
been deleted and GroEL is expressed exclusively from a plasmid-borne copy of
the
gene which can be tightly regulated by the arabinose PBS promoter and its
regulatory
gene, araC (Gunman, L.-M., Belin, D., Carson, M. J. & Beckwith, J. (1995) J.
Bacteriol. 177, 4121-4/30). AraC protein acts as either a repressor or an
activator
depending on the carbon source used. PBS is activated by arabinose but
repressed by
glucose. The AI90[pBAD-EL] cells can not grow on medium supplemented with
glucose at 37°C. None of the minichaperones are able to suppress this
groEL growth
defect.
We then determine whether the constructs can supplement low levels of GroEL
from
transfected AI90(pBAD-EL] cells by progressively switching on the plasmid-
borne
groEL gene by increasing arabinose from 0 to 0.2 % (Fig. 5). In the absence of
arabinose, the only cells that form colonies are those carrying pJC expressing
GroEL.
About 80% of the cells form colonies compared to the number produced in the
presence
of 0.2 % arabinose, at which concentration all cells are fully induced ( 100 %
viability) .
At 0.01 % arabinose, cells transfected with pJC expressing sht alone, sht-
GroEL(191-
376), sht-GroEL(191-345) or sht-GroEL(193-335)(Y203E) show little colony
forming
ability. Those containing pJC[sht-GroEL(193-335)], however, produced 15-20% of
the
number produced in the presence of 0.2% arabinose. At 0.07% arabinose, cells
containing pJCGroEL produce as many colonies as those fully induced by 0.2 % ;
pJCsht-GroEL[193-335] 75-80%; pJCsht-GroEL(i91-345]; 30-40%; pJCsht-
GroEL[I91-376] --20%; and the two controls, pJCsht-GroEL[193-335(Y203E)] and
pJCsht, -15 % . Thus, pJCsht-GroEL[193-335] can significantly supplement
depleted
levels of GroEL.
Example 4
Effect on ~, replication of over-expressing GroEL or the minichaperones.
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Heat induction of the groE operon has been shown to decrease burst size of 7~
bacteriophage in E. toll (Wegrzyn, A., Wegrzyn, G. & Taylor, K. (1996)
Virology
217, 594-597). In contrast, we have found that the over-expression of GroEL
alone
prevents plaque formation by ~, in wild-type strains including SV1. This
effect is
5 specific since neither over-expression of GroES alone or together with GroEL
causes a
significant drop in number of plaques formed. No effects on plaque counts from
over-
expressing the different minichaperones are found. Although the groE operon is
named for its effects on the E protein of 7~ (Georgopoulos, C., Hendrix, R.
W.,
Casjens, S. R. & Kaiser, A. D. (1973) J. Mol. Biol. 76, 45-b0), it seems that
the main
10 effect of GroEL over-expression is mediated through the ~, origin.
Both GroEL and the minichaperones inhibit replication of the Lorist6 plasmid
which
uses the bacteriophage ~, origin. A series of cultures of TG1 carrying
Lorist6,
encoding kanamycin resistance, and each of the pBAD30 vectors are titered on
LB
15 plates containing kanamycin and ampicillin in presence or absence of 0.2 %
arabinose,
which induces expression from the PBS promoter (Fig. 6). The percentage of
cells
forming kanamycin resistant colonies in presence of arabinose compared to the
number
formed in the absence of arabinose (100%) is shown for each of the expression
vectors.
Loss of kanamycin resistance reflects inhibition of Lorist6 replication. Over-
expression
20 of GroES alone has no effect, while GroEL decrease the number of kanamycin
resistant
colonies 200-fold (Fig. 6). Each of the minichaperones inhibits Lorist6
replication;
kanamycin resistant colonies are decreased by 10- to 23-fold (Fig. 6).
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
S
(i) APPLICANT:
(A) NAME: MEDICAL RESEARCH COUNCIL
(B) STREET: 20 PARK CRESCENT
(C) CITY: LONDON
1Q (E) COUNTRY: UK
(F} POSTAL CODE (ZIP): W1N 4AL
(ii) TITLE OF INVENTION: CHAPERONE FRAGMENTS
LS (iii) NUMBER OF SEQUENCES: 10
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AGCTTAACGG CCGTTG 16
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Met Arg Gly Ser His His His His His His Gly Leu Val Pro Arg Gly
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Met Ala Ala Lys Asp Val Lys Phe Gly Asn Asp Ala Arg Val Lys Met
1 5 10 15
Leu Arg Gly Val Asn Val Leu Ala Asp Ala Val Lys Val Thr Leu Gly
25 30
Pro Lys Gly Arg Asn Val Val Leu Asp Lys Ser Phe Gly Ala Pro Thr
35 40 45
z5 Ile Thr Lys Asp Gly Val Ser Val Ala Arg Glu Ile Glu Leu Glu Asp
50 55 60
Lys Phe Glu Asn Met Gly Ala Gln Met Val Lys Glu Val Ala Ser Lys
65 70 75 80
Ala Asn Asp Ala Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala
85 90 95
Gln Ala Ile Ile Thr Glu Gly Leu Lys Ala Val Ala Ala Gly Met Asn
100 105 110
Pro Met Asp Leu Lys Arg Gly Ile Asp Lys Ala Val Thr Ala Ala Val
115 120 125
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Glu Glu Leu Lys Ala Leu Ser Val Pro Cys Ser Asp Ser Lys Ala Ile
130 135 140
Ala Gln Val Gly Thr Ile Ser Ala Asn Ser Asp Glu Thr Val Gly Lys
145 150 155 160
Leu Ile Ala Glu Ala Met Asp Lys Val Gly Lys Glu Gly Val Ile Thr
165 170 175
Val Glu Asp Gly Thr Gly Leu Gln Asp Glu Leu Asp Val Val Glu Gly
180 185 190
Met Gln Phe Asp Arg Gly Tyr Leu Ser Pro Tyr Phe Ile Asn Lys Pro
195 200 205
Glu Thr Gly Ala Val Glu Leu Glu Ser Pro Phe Ile Leu Leu Ala Asp
210 215 220
Lys Lys Ile Ser Asn Ile Arg Glu Met Leu Pro Val Leu Glu Ala Val
225 230 235 240
Ala Lys Ala Gly Lys Pro Leu Leu Ile Ile Ala Glu Asp Val Glu Gly
245 250 255
Glu Ala Leu Ala Thr Leu Val Val Asn Thr Met Arg Gly Ile Val Lys
260 265 270
Val Ala Ala Val Lys Ala Pro Gly Phe Gly Asp Arg Arg Lys Ala Met
275 280 285
Leu Gln Asp Ile Ala Thr Leu Thr Gly Gly Thr Val Ile Ser Glu Glu
290 295 300
Ile Gly Met Glu Leu Glu Lys Ala Thr Leu Glu Asp Leu Gly Gln Ala
305 310 315 320
Lys Arg Val Val Ile Asn Lys Asp Thr Thr Thr Ile Ile Asp Gly Val
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325 330 335
Gly GluGlu AlaAlaIle GlnGlyArgVal AlaGlnIleArg GlnGln
340 345 350
Ile GluGlu AlaThrSer AspTyrAspArg GluLysLeuGln GluArg
355 360 365
VaI AlaLys LeuAlaGly GlyValAlaVal IleLysValGly AlaAla
370 375 380
Thr GluVal GluMetLys GluLysLysAla ArgValGluAsp AlaLeu
385 390 395 400
1$ His AlaThr ArgAlaAla ValGluGluGly ValValAlaGly GlyGly
405 410 415
Val AlaLeu IleArgVal AlaSerLysLeu AlaAspLeuArg GlyGln
420 425 430
Asn GluAsp GlnAsnVal GlyIleLysVal AlaLeuArgAla MetGlu
435 440 445
Ala ProLeu ArgGlnIle ValLeuAsnCys GlyGluGluPro SerVal
450 455 460
Val AlaAsn ThrValLys GlyGlyAspGly AsnTyrGlyTyr AsnAla
465 470 475 480
3~ Ala ThrGlu GluTyrGly AsnMetIleAsp MetG1yIleLeu AspPro
485 490 495
Thr LysVal ThrArgSer AlaLeuGlnTyr AlaAlaSerVal AlaGly
500 505 510
Leu MetIle ThrThrGlu CysMetValThr AspLeuProLys AsnAsp
515 520 525
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Ala Ala Asp Leu Gly Ala Ala Gly Gly Met Gly Gly Met Gly Gly Met
530 535 540
Gly Gly Met Met
545
