Note: Descriptions are shown in the official language in which they were submitted.
2 0 ~ 7 2 3 3
-- 1
D e s c r i p t i o n
The invention concerns a process for the enzymatic
treatment of substrates.
In virtue of their substrate specificity, enzymes are
extensively used in biotechnology and in diagnostics. In
biotechnology enzymes are used for the specific
conversion of substrates. Thus, derivatives of starch
can be produced by treatment with amylase. In
diagnostics specific conversions are carried out using
enzymes which lead to a signal which can be evaluated.
Moreover in diagnostics it is sometimes necessary to
specifically convert substrates before their detection.
Thus, for example, for the cholesterol determination
cholesterol esters present in the sample must be
saponified beforehand. For many of these applications it
is advantageous to immobilize enzymes. As a rule, this
is done by binding enzymes to a carrier material. In
this process, the binding of the enzymes is usually
covalent via spacers which are bound to functional
groups of the enzyme. A problem in this is that the
binding must be carried out in such a way that the
enzymatic properties of the enzyme are not impaired by
the immobilization and moreover that the binding is
carried out so that the active centre remains accessible
to the substrate. In addition, the binding of the enzyme
should be such that no detachment occurs under the
conditions during the reaction with the substrate.
Enæymes that are used for the above-mentioned purposes
are often produced by means of genetic engineering. For
this a gene coding for the desired enzyme is inserted
into a plasmid, and transformed and expressed in a
- 2 - ~V ~ 12 3 ~
suitable organism. The enzymes are isolated from the
lysate after cell lysis. The purification of the enzyme
often causes problems since very many proteins are
present in the lysate which is obtained and for further
processing it is absolutely essential that these are
separated.
It was therefore the object of the invention to provide
a biocatalyser for the enzymatic treatment of substrates
in which the enzymes in pure form are bound in a simple
manner and in such a way that their activity is not
impaired and that they do not become detached under the
reaction conditions during the isolation of the treated
substrate and that, as a consequence thereof, the
biocatalyser is available for as many conversion cycles
as possible.
This object is achieved by a process for the enzymatic
treatment of substrates which is characterized in that
the substrate to be treated is brought into contact with
a biocatalyser which was obtained by combining, in order
to produce a fusion protein, a gene which codes for a
biologically active substance and a DNA fragment which
codes for a binding peptide which can interact with a
carrier material in order to produce a fuslon protein,
thcn inserting the combined gene and DNA fragment into a
suitable vector, transforming it in a suitable organism,
culturing the organism, lysing the cells and bringing
the lysate into contact with a carrier material which is
capable of binding the binding peptide whereby the
fusion protein binds to the carrier material by
intermolecular interactions and the enzymatically
treated substrate is subsequently isolated.
~0~72~
-- 3
In order to produce the biocatalyser according to the
present invention, the enzyme in the form of a fusion
protein is immobilized by intermolecular interactions on
a carrier material in a non-covalent binding in such a
manner that the binding is not broken under the
conditions of the substrate conversion and as a result
of which the immobilized enzyme is available for further
reaction cycles. Several advantages are achieved by
binding via a peptide sequence fused to the N- or C-
terminal end of the enzyme. The binding does not impair
the active centre or the mobility~of the enzyme. In this
way the accessibility of the immobilized enzyme to the
substrate is improved. In addition, a purification is
not necessary since the enzyme can be bound directly
from the lysate to the carrier material.
Moreover, the immobilized enzyme can be detached from
the carrier material in order to regenerate the
biocatalyser in a manner known to the expert and the
biocatalyser can be again loaded with lysate containing
the enzyme without having to empty the bioreactor vessel
or dispose of the carrier material.
According to the present invention a biocataly~er is
used for the enzymatic treatment of substrates which
contains a carrier material to which a fusion protein is
bound via intermolecular interactions. This fusion
protein consists of the enzyme which i5 necessary for
the process and a binding peptide which mediates the
binding to the carrier material. All enzymes that can be
produced by genetic engineering are suitable for the
process accordiny to the present invention such as e.g.
~-glucosidase, glucose oxidase, aminoacylase, glucose
isomerase, creatinase, ~-galactosidase, pullulanase,
trehalase, trehalose phosphorylase, glucose
~7233
-- 4 --
dehydrogenas~, mannitol dehydrogenase, D-amino acid
oxidase (D-AOD), aldolase, cholesterol esterase, alcohol
dehydrogenase (ADH), pig liver esterase, subtilisin,
dehalogenases, naphthaline dioxygenase, chymotrypsin,
~-amylase and thermo-stable amylases, ligninase, nitrile
hydratase, horse radish peroxidase. Amino acid sequences
that can be produced by genetic engineering and which
can interact with a carrier material are suitable as the
binding peptide. The fusion protein can be produced
according to known molecular biological processes (T.
Maniatis, E.F. Fritsch and Sambrook, J. Molecular
Cloning, 1982, Cold Spring Harbor Laboratory). For this,
DNA fragments coding for the enzyme and the binding
peptide are inserted into a suitable vector. The vector
i9 then transformed in a suitable organism and, after
selection, this organism is then cultured in the usual
manner. The cultured cells are lysed according to known
processes and the lysate obtained in the lysis which
contains the fusion protein is brought into contact with
the carrier material. The fusion protein binds to the
carrier material via the binding peptide and can thus be
separated from the other substances contained in the
lysate.
A material which can interact intermolecularly with the
binding peptide is used as the carrier material.
Intermolecular interactions which are suitable for
immobilizing the fusion protein on the carrier material
are ionic, hydrophilic interactions, complex formation,
hydrophobic interactions, binding peptide/receptor
interactions as well as signal peptide/membrane
interactions. The carrier material and sequence of the
binding peptide are therefore chosen so that they can
participate in one of these interactions. Proteins and
carrier materials which are suitable for this are known
~ ~ ~ 7 2 3 ~
to the expert. The binding preferably takes place via
ionic interaction whereby the carrier material as well
as the binding peptide of the fusion protein have
charged groups or via the formation of complexes such as
those used e.g. in metal-chelate affinity
chromatography.
Gels such as those which are also used for
chromatography or ion-exchange resins are preferably
employed as the carrier materials. Carriers which can be
mechanically stressed are particularly suitable, in
particular hydrophilic polymerisates denoted fractogels
and matrices denoted "tentacle-type" gels which carry
exchanger centres on "tentacle-like" polymer chains
bound to a hydrophilic matrix. Other materials are also
suitable in a corresponding derivatised form such as
e.g. those materials denoted soft gels which are based
on polysaccharides and are well known for chromatography
such as dextrins, agarose or sepharose. For example
S-Sepharose~ ff~Pharmacia/LKB)
CM-Sepharose~ ff (Pharmacia/LKB)
SP-Sephade ~ C-50 (Pharmacia/LKB), for batch application
SE-EUPERGI ~ (R~hm Pharma)
Bio-Re ~ 70 (Bio-Rad)
Heparin-Sepharose~ CL-6B
Fractogel~ TSK SP-650 (Toyo Soda/Merck)
Fractogel~ EMD SO3 -650 (Merck, "tentacle-type gel")
Q-Sepharose~ ff (Pharmacia/LKB)
DEAE-Sepharose~ ff (Pharmacia/LKB)
DEAE-Sephade ~ A-50 (Pharmacia/LKB), for batch
application
QAM-EUPERGIT~ (Rohm Pharma)
AG MP-l (Bio-Rad)
Fractogel~ TSK DEAE-650 (Toyo Soda/Merck)
Fractogel~ EMD TMAE-650 Merck, "tentacle-type gel")
- 6 - ~ 2 3 ~i
can be used as derivatives. Fractogel~ EMD S03 -650 is
preferably used as the carrier material when the binding
peptide contains arginine and lysine as amino acids.
When glutamic acid and aspartic acid are used for the
binding peptide, Fractogel~ EMD TMAE-650 is preferably
used.
Chelate-forming resins such as e.g. TSK chelates 5-PW or
chelating Sepharose~ 6B ff which have iminodiacetate as
ligands, as well as tris-(carboxy~methyl)-
ethylenediamine-agarose are suitable for binding the
fusion proteins. A column material is particularly
preferred as the carrier material which contains the
metal chelator nitrilo-triacetic acid (NTA) as the
ligand which mediates the binding to the fusion protein.
The protein sequence of the fusion protein which is
responsible for the binding then contains poly-
histidine. The binding then takes place via carrier-
bound metal ions via formation of a complex with the
functional histidine residues.
A carrier material is particularly preferably used which
has negatively or positively charged groups. The protein
sequence of the fusion protein which is responsible for
the binding then has oppositely charged group~ which can
be introduced by the amino acids lysine and/or arginine
or glutamic acid and/or aspartic acid. The binding then
takes place via polyionic interactions.
The binding peptide part of the fusion protein i5
composed of several amino acids, whereby the length of
the peptide is chosen so that a sufficiently strong
binding of the fusion protein to the carrier material is
ensured and so that the active sites of the enzyme are
accessible for substrate binding. The binding peptide is
3 j
preferably composed of 2 to 30 amino acids. In a
preferred embodiment the binding peptide is not only
composed of the amino acids which have the functional
groups which are necessary for the binding to the
carrier material but contains in addition several other
amino acids which improve the accessibility of the
enzyme. The introduction of a proline or glycine polymer
with 1 to 10 amino acid groups is particularly suitable.
It is expedient if the amino acid sequence of the
binding protein is so chosen that it is substantially
resistant to digestion by proteinases.
The binding peptide does not have to be composed of a
homogeneous chain of the same amino acids. It can also
have a combination of similarly charged amino acids. The
binding peptide is preferably composed of a sequence
which contains the amino acids arginine and/or lysine or
aspartic acid and/or glutamic acid.
In order to protect the binding peptide part of the
fusion protein against proteolytic digestion the free
end of the binding peptide is preferably protected by an
amino acid such as e.g. proline which is fused to it and
that is not charged and is not easily accessible to
exoproteinases.
Since the enzyme contalned in the fusion protein can be
present in a partially denatured form as a result of the
processing and cannot in this case develop its full
activity, in a preferred embodiment the enzyme is
renatured according to well-known methods after
immobilizing it on a carrier in which it is first
treated with a denaturing agent and subsequently a
renaturation step is carried out. The conditions for
this are adjusted so that no detachment of the fusion
~72~.~
protein can take place. This has the particular
advantage that reaggregation, which would otherwise be a
risk in the renaturation, is not possible because of the
immobilization.
In order to bind the fusion protein to the carrier
material, the lysate obtained after cell lysis is
brought into contact with the carrier material under
conditions which favour the binding. In this process the
binding peptide binds via its functional groups to the
corresponding functional groups of the carrier material.
The biologically active immobilized enzyme obtained in
this way can then be used for the treatment of
substrates. The substrate is brought into contact with
the immobilized enzyme in the usual manner and
subsequently the enzymatically treated substrate is
isolated. The immobilized enzyme used according to the
present invention is preferably filled in a column and
the substrate is passed over it in a well-known manner.
A further object of the invention was to provide solid
phase-bound or particle-bound, specifically bindable
peptide and protein substances (receptors) for use in
enzyme-immunoassays in which the receptors are bound in
a pure form in a simple manner in such a way that their
immunological activity is not impaired and a detachment
under the reaction conditions during the immunoassay
procedure does not take place.
This object is achieved by a method for the detection of
specifically bindable substances according to the
immunoassay principle using a solid phase-bound receptor
which is characterized in that the sample solution as
well as at least one labelled receptor which is capable
of binding to the substance to be detected are brought
~ 3
_ g
into contact with a solid phase-bound receptor which was
obtained by combining, in order to produce a fusion
protein, a gene which codes for a biologically active
substance and a DNA fragment which codes for a binding
peptide which can interact with a carrier material then
inserting the combined gene and DNA fragment into a
suitable vector, transforming it in a suitable organism,
culturing the organism, lysing the cells and bringing
the lysate which contains the fusion protein into
contact with a carrier material which is capable of
binding the binding peptide whereby the fusion protein
binds to the carrier material by intermolecular
interactions and the after incubation the solid phase is
separated from the liquid phase and the label is
determined in one of the two phases.
A method for the detection of specifically bindable
substances according to the principle of the homogeneous
immunoassay using a particle-bound receptor is provided
as a further embodiment which is characterized in that
the sample is incubated with a particle-bound receptor
which is capable of binding to the substance to be
detected, which receptor was obtained by combining, in
order to produce a fusion protein, a gene which codes
for a biologically active substance and a DNA fragment
which codes for a binding peptide which can interact
with a carrier material then inserting the combined gene
and DNA fragment into a suitable vector, transforming it
in a suitable organism, culturing the organism, lysing
the cells and bringing the lysate which contains the
fusion protein into contact with a carrier material
which is capable of binding the binding peptide whereby
the fusion protein binds to the carrier material by
intermolecular interactions and after incubation the
3 ~
-- 10 --
solid phase is separated from the liquid phase and the
agglutination is determined after incubation.
According to the present invention the solid phase-bound
or particle-bound receptor used is immobilized in the
form of a fusion protein by intermolecular interactions
on a carrier material in a non-covalent binding in which
the carrier material can be present either as a solid
phase e.g. in the form of tubes, microtitre plates or
polystyrene beads or in the form of particles e.g. latex
particles. The fusion protein consists of an
immunologically active substance which is necessary for
the method and a binding peptide which mediates binding
to the carrier material. All immunologically active
substances which can be produced by genetic engineering
e.g. antibodies and their fragments as well as antigens,
e.g. HIV or hepatitis antigens or haptens such as
hormones, drugs etc. are suitable for the method
according to the present invention. The same peptides as
described previously are suitable as the binding
peptide. A material which is suitable for use in an
immunoassay as a solid phase or as a particle and which
can interact intermolecularly with the binding peptide
i9 used as the carrier material . Carrier materials
whiah are suitable for this are known to the expert. The
process for the production of the fusion protein i8
carried out as described above. ~he solid phase-bound or
particle-bound receptors produced in this way can be
used for immunoassays of the heterogeneous and
homogeneous type in a well-known manner. The procedure
for these immunoassays is known to the expert and does
not need to be described here in detail. Basically in
this process the sample solution is reacted with at
least two receptors whereby in the case of the
heterogeneous immunoassay one receptor is bound to a
~ V ~ a
solid phase and the other is labelled and in the case of
the homogeneous immunoassay one receptor is particle-
bound and the other can bind to the substance to be
detected and to the particle-bound receptor. Complexes
form between the receptors and the substance to be
detected which result in a change in signal which, in
the case of the heterogeneous immunoassay, is due to the
label which can be an enzyme, or a fluorescent,
chemiluminescent or radioactive substance and, in the
case of the homogeneous immunoassay, is due to
agglutination.
The invention is elucidated by the following figures and
examples:
Fig. 1 shows the construction of the ~-glucosidase-Arg6
expression vector;
Fig. 2a shows the plasmid map of pKK177-3/GLUCPI_ARG6;
Fig. 2b shows the nucleotide sequence of the plasmid
pKK177-3/GLUCPI ARG6;
Fig. 3 shows a diagram of a maltose biocatalyser with
complete substrate conversion; substrate buffer:
10 mM KPP, 1 mM EDTA, 6 mM maltose, pH 7.0;
Fig. ~ shows a diagram of a maltose blocatalyser with
substrate conversion limited by substrate
saturation
substrate buffer: 10 mM KPP, 1 mM EDTA, 0.15 M
maltose, pH 7.0;
Fig. 5 shows the renaturation kinetics of
~-glucosidase-Arg6 denatured on a carrier
Denaturation buffer: 10 mM KPP, 1 mM EDTA, 2 mM
DTE, 6 M urea, pH 6.8
7 ~ ~ 3
Renaturation buffer: 10 mM KPP, 1 mM EDTA,
0.15 M maltose, pH 6.8 .
E x a m p l e s
Standard methods wer~ used to manipulate DNA such as
those described by Maniatis et al., (1982) in Molecular
Cloning, Cold Spring Harbor Laboratory, Cold Spring
Harbor, New York 11724. The molecular biological
reaqents which were used were employed according to the
manufacturers' instructions.
Materials:
Restriction endonucleases and T4-DNA ligase were from
Boehringer Mannheim GmbH; yeast extract, Bacto Trypton
and Casamino acids were from Difco.
~acterial strains:
The E. coli strain HB101 (Maniatis et al., 1982,
Molecular Cloning: A laboratory manual. Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, New York)
or GM~8 (Yanisch-Perron et al., 1985, Gene 33, 103-119)
for the preparation of unmethylated DNA, were used as
recipient strains for the plasmid amplification. The
E. coli strain RM82, a methionine revertant of ED8654,
(Murray et al., 1977, Mol. Gen. Genet. 150, 53-61),
which contains (i) the R-plasmid pREM6677 (pePA119,
DSM 3691P) which co~es for the lacIq repressor and for
trimethoprime resistance and (ii) the ~-glucosidase PI-
Arg6 expression plasmid pKK177-3/GLUC ARG6 (ampicillin
resistance), was used for the expression of the
~-glucosidase-PI-Arg6 fusion protein. This strain is
denoted RM82 Iq/pKK177-3/GLUCPI_ARG6 in the following.
~ 3
E x a m p l e
Construction of the ~-glucosidase-Arg6 expression
plasmid pKK177-3/GLUCPI-ARG6
The structural gene of the ~-glucosidase PI from baker's
yeast was extended at the 3' end by a DNA fragment which
codes for the amino acid sequence GlyArgArgArgArgArgArg.
In this way an ~-glucosidase PI fusion protein forms
which contains 6 additional argini~e residues
(polycationic ankering sequence) and a glycine as a
"spacer" at the C-terminus.
For this purpose the ca. 4.7 kbp long plasmid pKK177-
3/GLUCPI (production and description cf: EP-A O 300 425,
Kopetzki, Schumacher, Buckel, 1989, Mol. Gen. Genet.
21~, 149-155) was partially digested with the
restriction endonuclease EcoRI and completely digested
with BclI. The synthetic DNA fragment
5'-AATTATGACGATATCC-3'
3'-TACTGCTATAGGCTAG-5'
M~tThrIleSer....
was ligated lnto the isolated ca. 4.7 kbp long
BclI/EcoRI vector fragment (construction: pKX177-
3/GLUCPI SD) which destroys the EcoRI and BclI
-
restriction cleavage site. The ca. 300 bp long EcoRI
fragment was isolated from the plasmid YRp/GLUCPI (DSM
4173P~ (production and description cf: EP-A O 323 838;
Kopetzki, Buckel, Schumacher, 1989, Yeast 5, 11-24).
This was re-cleaved with HinfI and the ca. 90 bp long
EcoRI/HinfI fragment was isolated.
~ O ~ !~7 2 ~ ~
-- 14 --
Afterwards the ca. 90 bp long EcoRI/HinfI fragment as
well as the synthetic DNA fragment
IleTyrLeuValLysGlyArgArgArgArgArgArgEndEnd
5'-AATCTACCTGGTCAAAGGGCGCCGACGTCGCCGGCGTTAATA
3'-GATGGACCAGTTTCCCGCGGCTGCAGCGGCCGCAATTATTCGA-5'
______ _____
HinfI HindIII
were inserted via a three-fold ligation into the
ca. 2.85 kbp long EcoRI/HindIII-pKK177-3 vector fragment
(DSM 3062) (construction: pKK177-3!EH).
The ca. 130 bp long EcoRI/HindIII fragment was isolated
from the plasmid pKK177-3/EH and ligated into the
ca. 4.65 kbp long EcoRI/HindIII-pKK177-3/GLUCPI-SD
vector fragment (construction: pKK177-3/GLUCPI ARG6).
The correct construction of the a-glucosidase-Arg6 gene
was confirmed by restriction analysis and DNA
sequencing. The construction oP the ~-glucosidase-Arg6
expression Vector is shown diagrammatically in Figure 1.
In order to express the ~-glucosisase-Arg6 fusion
protein, the plasmid pKKl77-3/GLUCPI ARG6 (Figure 2a and
2b, SEQ ID No:1) Was transformed into the E. coli strain
RM82 Iq (ED Iq, DSM 2102).
7 ~
- 15 -
E x a m p 1 e 2
Expression of the ~-qlucosidase-Arq6 fusion protein in
E. coli
The E. coli strain RM82 which has the ~-glucosidase-PI-
Arg6 expression plasmid and a R plasmid which codes for
the lacIq repressor as well as for trimethoprime
resistance was used for the expression of the
~-glucosidase-PI-Arg6 gene. The e~pression of the
~-glucosidase is under the control of the tac-hybrid
promoter.
Culture conditions
500 ml modified M9 minimal medium (6 g Na2HPO4/1, 3 g
KH2P04/1, 0.5 g NaCl/l, 1 g NH4Cl/1, 2.5 mg thiamine/l,
5 g Casamino acids/l, 20 ml glycerol/l ~87 %), 0.25 g
MgSO4 7 H2O/1, 50 mg ampicillin/l, 10 mg
trimethoprime/l) was inoculated with 1 % of an overnight
culture of the E. coli strain RM82-Iq/pKK177-
3/GLUCPI ARG6 (which was also in the above-mentioned
medium). The cultures were incubated at 30C while
shaking constantly. The growth was monitored by
measurement of the optic~l density at 550 nm (OD550nm).
Induction of the ~-glucosidase
After a cell density of OD550nm 0.7 - 0.9 was reached
the formation of ~-glucosidase was induced by the
addition of lactose (final concentration 0.5 %). The
cells were harvested by centrifugation after ca. 16
hours.
~ s 2 ~3~
- 16 -
The increase in the ~-glucosidase content in the cells
was monitored with the aid of 1 ml culture samples which
were taken at different times after the induction and
analysed.
Determination of the ~-qlucosidase activitv
The cell pellet from 1 ml E. coli culture was
resuspended in O.5 ml 10 mM phosphate buffer, 1 mM EDTA,
pH 7.0, the cells were lysed by m,eans of ultra sound,
the cell debris was separated by cell centrifugation and
the supernatant was processed further as the cell
lysate.
50 ~l of the supernatant (dilute if necessary~ was mixed
with 3 ml potassium phosphate buffer (lOOmM, pH 6.8)
which contained the ~-glucosidase substrate
p-nitrophenyl-~-D-glucopyranoside (p-NPG; 2 mM). The
~-glucosidase liberates p-nitrophenol by enzymatic
cleavage. This was determined by the increase of
absorbance at ~05 nm.
E x a m p 1 ~ 3
P~oductio~ of th~ b~oç~talyser
Cell lYsis and isolation of the crude extract
The cell pellet from 500 ml E. coli culture was
resuspended in 30 - 50 ml potassium phosphate buffer
(KPP), l mM EDTA, pH 7.0, mechanically lysed by means of
a French press (2 passages at 14000 psi; 1 psi= 0.068
atm) and subsequently the cell debris fragments were
' 2 3 ~
- 17 -
separated by centrifugation (10 min, 7000 rpm). The cell
lysate obtained in this way was used directly for the
production of the biocatalyser.
carrier materlals
The cation-exchanger Fractogel~ EMD S03 -650 ("tentacle-
type gel") was used whose exchanger groups are freely
mobile in space on flexible polymer chains. By this
means a good interaction of the c~lumn material with the
proteins to be bound is ensured.
The cation-exchanger Fractogel~ EMD S03 -650 was
eguilibrated with 10 mM potassium phosphate buffer, 1 mM
EDTA, pH 7Ø As an alternative heparin-Sepharose~ CL-6B
was used as the gel matrix. The binding capacity for the
~-glucosidase-Arg6 fusion protein was determined as 3000
U/ml for both gel materials.
Determination of the extent of loading
In order to immobilize the ~-glucosidase-Arg6 fusion
protein on Fractogel~ EMD S03 -650 or Heparin-Sepharose~
CL-6B, the crude extract (a-glucosidase activity ca.
200 U/ml) was applied to the column at a pumping rate of
ca. 10 column volumes/hour. Afterwards the column was
washed with 10 mM potassium buffer, 1 mM EDTA, pH 7.0
and the loading of the column material with ~-
glucosidase-Arg6 fusion protein was determined. For this
the total activity of ~-glucosidase which was not bound
to the column was measured in the column eluate (eluent
and washing buffer) and subtracted from the amount of
enzyme applied to the column. The binding of the
~7~3~
- 18 -
~-glucosidase-Arg6 fusion protein to Fractogel~ SO3 -650
or heparin-Sepharose~ CL-6B was 95-98 %.
E x a m p l e 4
Conversion of substrate
In order to test the function of the ~-glucosidase
catalyser, the hydrolytic properties of the enyzme were
determined with respect to the ~-~lucosidase substrates
maltose and p-NPG. The amount of glucose released from
maltose, or the nitrophenol released from p-NPG, per
unit time by the a-glucosidase catalyser is a measure
for the catalyser performance. The release of
p-nitrophenol from p-NPG was determined photometrically
in the column eluate (analogous to the ~-glucosidase
determination). The hydrolysis of maltose was monitored
in the column eluate via the release of glucose.
Glucose determination
Test principle:
hexokinase
D-glucose ~ ~TP -~ > glucose-6-P + ADP
glucose-6-phosphate
dehydrogenase
glucose-6-P + NADP+ --------------> gluconate-6-P
+ NADPH+H+
6 2 3 ~
-- 19 --
Test procedure:
625 ~1 triethanolamine buffer (0.3 M TRA, 4 mM MgC12,
pH 7.5)
+ 50 ~1 ATP/NADP solution (150 mM ATP, 12 mM NADP in the
above-mentioned TRA buffer)
+ 50 ~1 sample containing glucose
Read the absorbance at 366 nm (A1)
+ 10 ~1 enzyme mixture (glucose-6-phosphate
dehydrogenase
1 mg/ml and hexokinase 1 mg/ml; each in 3.2 M ammonium
sulphate solution).
After the reaction has reached completion A2 is read at
366 nm.
The glucose content of the sample is calculated from the
difference A2 ~ A1.
Maltose hydrolysis by means of non-covalently
immobilized a-alucosidase
Maltose bioreactor with çomplete substrate conversion
2500 U ~-glucosidase-Arg6 was immobilized on a heparin-
Sepharose~ CL-6B column (ca. 3 ml column volume)
according to the procedure described in Example 3. The
substrate buffer contained 6 mM maltose; the pumping
rate was 0.5 ml/min.
In continuous operation, the hydrolysis of maltose was
observed over a time perîod of 33 days (Figure 3).
Result: The reactor showed 100 ~ substrate conversion
during the entire period of observation.
~ o a~ r~ 2 -~ ~
- 20 -
Maltose bioreactor with limited substrate conversion
180 U ~-glucosidase-Arg6 was immobilized on a Fractogel~
EMD S03 -650 column as described in Example 3. In order
to compare the substrate conversion and the binding
stability at different temperatures, a reactor was
operated at room temperature (RT) and a further reactor,
which was loaded identically, was operated at 30C.
The substrate buffer contained 0.15 M maltose; the
pumping rate was 5 column volumes/hour. The maltose
conversion of the bioreactor was observed over 30 days.
During this time period a decrease from initially 50 %
to 38 % (30C) or from 36 % to 28 % (RT) was observed
(Figure 4).
E x a m p l e 5
Renaturation of denatured ~-glucosidase-Arq6 fusion
prote~ on the carrier
Purified a~glucosidase-Arg6 fusion protein (ca. 80 U/mg)
was denatured in 10 mM potassium phosphate buffer, 1 mM
EDTA, 6 M urea, 2 mM DTE pH 7.0 and applied to a
"tentacle-type gel" column equilibrated with denaturing
buffer. After application of the sample, the column was
rinsed with renaturing buffer (lO mM potassium phosphate
(KPP), 1 mM EDTA, 2 mM 1,4-dithioerythritol (DTE),
pl~ 7.0) (ca. 10 column volumes). The bound and renatured
~-glucosidase-Arg6 fusion protein was eluted with 10 mM
potassium phosphate buffer, 1 mM EDTA, 1 M NaCl, pH 7Ø
In the eluate obtained with 1 M NaCl, a specific
~-glucosidase activity of ca. 20 U/mg could be detected.
~ii 6123
~ x a m p l e 6
De- and renaturation of native ~-alucosidase-Ara6 bound
to the carrier
The carrier ma~erial Fractogel~ EMD SO3--650 was loaded
in a batch procedure with purified ~-glucosidase-Arg6
fusion protein (ca. 800 U), filled in a column, rinsed
for one hour with denaturing buffer (10 mM KPP, 1 mM
EDTA, 2 mM DTE, 6 M urea, pH 6.8) and subsequently
washed with substrate buffer (10 mM KPP, 1 mM EDTA,
0.15 M maltose, pH 6.8).
The renaturation of the ~-glucosidase can be monitored
in the column eluate by the increase in the glucose
released from maltose (Figure 5).
E x a m p 1 e 7
Immunoloqical determination of anti ~IV antibodies usina
an antiqçn bound to a solid ph~se
HIV ~nti~~n
The HIV polypeptide HIV2(envgp32)-~IVl(poly32-envgp41-
gagpl7-p24-15-poly(Arg-Lys) is a "multifunctional" HIV
fu.sion polypeptide produced by means of recombinant DNA
technology. It consists of protein partial regions of
the gag, pol and env region of HIVl and of the env
region of the HIV2 retrovirus. In addition the HIV
polypeptide has at its C-terminal a polycationic anchor
sequence consisting of 13 positively charged amino acids
(arginine and lysine).
~A7~3~
- 22 -
The protein sequence of the HIV polypeptide is shown in
SEQ ID NO:2.
Expression of the HIV fusion protein in E. coli
The E. coli strain RM82+, DSM 5446 (a lactose revertant
of RM82) which contains the HIV fusion protein
expression plasmid pKK233-2/MYYL_gp32_polp32_gp41_p24-
polyArgLys (ampicillin resistance) and the dnaY-lacq
plasmid pUBS500 was used for the expression of the HIV
fusion protein.
The construction of the HIV expression plasmid is
described in the German Patent Application P 40 02 636.1
dated 30.01.1990.
pUBS500 (EP-A 0 373 365) is a pACYC derivative (Chang
and Cohen, J. Bacteriol. 134 (1978) 1141-1156) which in
addition to the kanamycin resistance gene has a lacIq
repressor gene (Carlos, Nature 274 (1978) 762-765) and a
gene for the t-RNA arginine (anticodons: AAG, AGG) which
is rare in E. coli.
The recombinant E. coli cells were cultured at 30C in
DYT medium (16 g bactotryptone, 10 g yeast extract, 5 g
NaCl per liter) supplemented with 50 mg/l ampicillin and
50 mg/l kanamycin. After reaching an optical density of
0.6 - 0.8 at 550 nm the cells were induced with IPTG
(isopropyl-~-D-thiogalactopyranoside, final
concentration 1 mM). After an induction time of 4 - 10
hours the cells were centrifuged, washed with 10 mM
Tris-HCl buffer, pH 7.0 and stored at -20C until
further processing.
~B~72~3
- 23 -
Cell lysis, solubilization of the HIV fusion proteln
20 g (wet weight) E. coli cells were resuspended in
400 ml 100 mM Tris-HCl buffer, pH 6.5 - 7.5, 0.25 mg/ml
lysozyme was added and incubated for 0.5 - 1 hour at
room temperature. Afterwards the suspension was cooled
to 0 - 4C and the cells contained therein were lysed by
ultrasonication (French press). The cell debris and the
insoluble aggregated HIV fusion protein ("inclusion
bodies") were separated by centrifugation and the pellet
was washed 1 - 2 times with 200 - 400 ml 0.5 M NaCl or
KCl and 1 ~ (v/v) Triton-X-100. Subsequently the pellet
was resuspended in 20 ml 50 mM Tris-HCl pH 8.0
containing 8 M urea and 5 mM ~-mercaptoethanol at room
temperature for 1 hour while stirring (magnetic stirrer)
and the insoluble cell components were removed by
centrifugation. The protein concentration of the
solubilized proteins was determined according to
Bradford (Anal. Biochem. 72 (1976) 248-254) modified
according to Gotham et al. (Anal. Biochem. 173 (1988)
353-358).
~IIVl (polp32-~nvqp41-q~qP17-p24-15-poly¢~q-L~s) protei~
on a ~el mat~l~
1 ml cation exchanger Fractogel~ EMD-S~3-650(M)
equilibrated in 50 mM Tris-}lCl, pH 8.0 and 8 M urea was
incubated with 100 - 200 mg solubilized HIV fusion
protein at room temperature for 2 - 6 hours while
shaking. The proteins which were not bound to the gel
were removed by washing with equilibration buffer.
Afterwards the gel was re-incubated with 10 mg/ml bovine
serum albumin in phosphate-buffered saline (PBS, 0.15 mM
~723-~
- 24 -
sodium phosphate; 0.9 % NaCl; pH 7.2) and washed with
demineralized water containing 0.5 % Tween 20.
Determination of anti HI~ antibodies
The sample (10 - 100 ~l human serum) was diluted one
hundred-fold in PBS containing 10 % calf serum and
incubated for 4 - 12 hours at 37C with 10 ~l gel which
was coated with HIV fusion protein. It was subsequently
washed three times with 1 - 1.5 ml washing solution
(0.5 % Tween 20 in demineralized water).
In the second step it was incubated with a conjugate of
peroxidase and polyclonal antibody (POD conjugate,
ca. 30 mU peroxidase/ml in PBS) which was directed
against the Fc ~part of human IgG and washed three times
with washing solution.
Afterwards 1.5 ml substrate solution (1.6 mM 2,2'azino-
di-[3-ethylbenzthiazoline sulfonic acid(6)]diammonium
salt (~BT ~ ; 95 mM phosphate-citrate buffer, pH 4.4;
3.1 mM sodium perborate) was added, incubated for 15 -
~0 min at room temperature and the absorbance in the
centrifuged sample was determined at 492 nm as a measure
for the amount of specific antibody present in the
sample.
Normal sera showed a relative signal strength in
relation to anti-HIV pool sera (oD492 anti-HIV pool
serum/OD49Z normal serum) of 7 - 50 (oD492: absorbance at
492 nm).
Details of the deposition of the aforementioned cell
lines are set forth below.
-- 2 5 -- i~ 7 2 3 ~
DSM DEUTSCHE SAMMLUNG VON MIKROORGANISMEN
German Collection of Microorganisms
GESELLSCHAFT FUR BIOTECHNOLOGISCHE FORSCHUNG MBH
DSM Grisebachstrasse 8 D-3400 Gottingen,Germany Tel.:t0551) 393822
393823
_
Boehringer Mannheim GmbH Date
Biochemica Werk Tutzing 7.5.1g86
Postfach 1263/64
8132 Tutzing
ACKNOWLEDGMENT OF RECEIPT
We acknowledge that the plasmid with the identification reference
pePa 119
given by the depositor was deposited on the 9.4.1986 in the German
Collection of Microorganisms under the receipt number DSM 3691P.
GERMAN COLLECTION
OF MICROORGANISMS
- 26 ~ 2 ~ ~3
DSM DEUTSCHE SAMMLUNG VON MIKROORGANISMEN
German Collection of Microorganisms
GESELLSCHAFT FUR BIOTECHNOLOGISCHE FORSCHUNG MBH
DSM Grisebachstrasse 8 D-3400 Gottingen,Germany Tel.:(0551) 393827
Boehringer Mannheim GmbH
Biochemica Werk Tutzing
Abt. E - B 1
Postfach 1263/64
8132 Tutæing
Your ref. Our ref. Date
38687 14.07.1987
ACKNOWLEDGMENT OF RECEIPT
We acknowledge that the plasmid with the following reference number
YRp-Gluc.pI
given by the depositor was deposited on the 29.06.1987 in the German
Collection of Microorganisms under the receipt number
DSM 4173 P.
GE~MAN CO~LECTION
OF MICROORGANISMS
- 27 - ~li 72 ja
DSM DEUTSCHE SAMMLUNG VON MIKROORGANISMEN
German Collection of Microorganisms
GESELLSCHAFT FUR BIOTECHNOLOGISCHE FORSCHUNG MBH
DSM Grisebachstrasse 8 D-3400 Gottingen,Germany Tel (0551)393827
Boehringer Mannheim GmbH
Biochemica Werk Tutzing
Abt. E - B 1
Postfach 1263t64
8132 Tutzing
Your ref. Our ref. Date
Bk-Bl 39087 14.7.1987
Use of a DSM strain in a patent application
Confirmation
We hereby confirm that the microorganism mentioned below is present in
the general collection of the DSM. The strain will be stored for at
least a further 30 years from the date of this confirmation. The term
is calculated from the date of publication if the strain was referred
to when the patent was applied for and the DSM was informed of this.
In addition the culture will be stored for a term of 5 years after the
last application for delivery of a sample has been received.
Samples of this culture which are capable of reproduction were and
will be issued to anyone during the entire period according to the
national regulations for trading with microorganism~.
An examination of the viability was carried out on the date mentioned
below. The micxoorganism was viable at this time.
Taxonomic designation DSM Date of Date of Date of patent
for the microorganism number accession viability publication
in the DSM test according to the
applicant
Escherichia coli HB101 1607 16.7.1979 9.3.1987
Micromonospora 43141 15.4.1975 10.7.1987
echinospora
German Collection
of Microorganisms
BUDAPEST TREATY OU THE INTERNATIONAL
~ 2 8 - R~COGNITIOH OF THE DEPOSIT OF MICROORGANISMS
COR THE PURPOSES OF PATENT PQOCEDURE ~ f~
INTERNAT~ONAL FORM
Boehringer Mannheim GmbH
Biochemica W~rk Tutzing
Abt. E--Bl RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
Postfach 1263/64 issued pursuant to Rule 7 1 by the
INTERNATIONAL DEPOSITARY AUTHORITY
D--8132 Tutzing identified at the bottom of this poge
~AME AND ADDRESS
OF DEPOSITOR
L J
1 IDENTIFICATION OF THE MICROORGANISH
~.......... .
Idont1fleation reforonce ~iven by the Accession number given by the
DEPSIToR BNTU INTERNATIONAL DEPOSITARY AUTHORlTr
54-2/pKK 177-3 DSM 3062
.
Il SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOHIC DESIGNATION
. __
The microor9ani8m ldontified under I abovo ~as accompanied by
) a sciontiflc dascrlption
tXl a propos d taxonomic do4i~natir~n
tH~rk ~ith a cro8s uare npp~icabla)
_ ~
Ill RECEIPT AND ACCEPTANCE
Thl~ Intornational D~poaitary Author'ty occ~pta tho microor~ni8m idQntitiQd under I obovo
uhlch ~a8 rocoivad by it on 2 6 - O 9 - 1 9 8 4 ~dat~ o~ th- or~g~nn p
._ _ ........ _ _ .. _ .
IV INTERNATIONAL OEPOSITARY AUT~ORITY
. .. . __ __ ~
NaT~ GERHAN COLLECTION Signatura~) ot p r~on~) hnving tha po~or
OF MICROORG~NISHS to roproaant tho Intarnatlonal Depository
Addrass Grl~abachntr~n- G Authorlty or ot authorl~ed o~tlclol~)
D_3400 Go~tlngan
. 3-10-1984
_ .
1 ~hero Rulo 6 4~d) opp~ies such dote is tho date on ~hich the status of international depositary authority ~as acquired
~here a doposit moda outsid- tho 8udapest Trenty ofter the acquisition of the status of international depositary authority
is converted into a deposit under the Budapest Treaty such date is the date on ~hich the microorganism ~as received by the
intern~tiona~ depositary outhority
Form BP/4 ~sole page) 1081
BUDAPEST TREATr ON TNE INTERNATIONAL
- 2 9 - RECOGNITION OF THE DFPOSIT OF MlCROORGANlSMs ~ O ~ ~ ~ 3
FOR THE PURPOSES OF PATENT PRocEDuRE
INTERHATIONAL FORM
Boehringer Mannheim GmbH
Postfach 120 RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT
issued pursuant to Rule 7 1 by the
8132Tutzing INTERNATIONAL DEPOSJTARY AuTHoRlTr
identified st the bottom of this page
NAME AUD ADDRESS
OF DEPOSITOR
L J
. . ~
I IDENTIFICATION OF THE MICROORGA~ISH
_ ,....... _ .
Identificstion reforoneo given by the ~ccossion numbor given oy tho
DEPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY
BMTU 2602
. DSM 2102
- _
Il SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOH C DESIGNATION
._
The microorgonism 1dent1fled undor I abovo ~D8 accomponied oy
) o se10ntifie doserlptlon
X) e proposed toxonomie designotion
~Hork ~1th o eros~ ~ore npplienblo)
~-- - ...
Ill RECEIPT AND ACCEPTANCE
_ _ ,_ __ _ . .
This Intornat10nnl Dopositnry Author1ty oecopt8 tho microor90nl8m Idantlt1Qd unchr I ooovo ;
uh~ch ~8 roco~vod by ~t on 1 - 1 0 - 1 9 8 1 ~cbt~ ot tho or~9~n~ d p ~ )
,_ . . ,_ . . .__ _ - _ _ _ _
IV INTERNATIONAL DEPOSITARY AUTNORITY
. _ .......... ,= ,,_ . _ . _ _ __
NQme~ GERHUN COLLECTION Si~noturo~) ot p rnon~) h~vlng tho po~-r
OF MICROORGANI5HS to reprosQnt tho Intornat10nol Dopo~ltnry
Addrosr Grlgooqeh~tro~no ~ Authority or of cuthor1~eci offic1al~s)
D_3400 GoLt1ngon
~ .C~t
~ Dnto 2-06-1982
. _
I ~horo Rulo 6 4(d) applies such date is the date on ~hich the stotus of international depositnry authority ~a~ acr1uireci;
~horo a deposit mado outsido the SudDpest Tre~ty ofter the acquisition of the status of internntional depositary authority
is convorteci into a deposit urder the Budepest Treaty such date is the date on which ~he microorganism ~as received by the
internotional depositary authority
Form BP/4 ~sole page) 1081
. . _ _ _ .
~ ~ l 7 2 ~,3
SEQ ID NO:1
TYPE OF SEQUENCE: Nucleotide sequence
LENGTH OF SEQUENCE: 4648
FORM OF STRAND: single strand
Plasmid Pkkl77-3/GLUCPI_ARG6
181 ~1~1~ G~a~ CA~ICG ~1~ (~1~ GCGG~CA
241 ~a ~a;c~ ~CGAT A~G~ (~GMACAG AZ~CC~
421 ~ ~1~1~ CGrm;~ Cl~l~CA~ CA.:Ga~ ~T
541 l~ Cl~ Gl~ CAI~ ~1~ ACCN~
661 Gll~l~l~ A ~ CC~ ~aG6~ A C ~ A ~ l~ C ~ AC~
721 Ir~r~LXTlr~F~XT~ GTn~qIG G~rcT~T GA~K~YPA ~DG~TTA
781 CCTCCG~ T~CG~GTC GDCh~T~A CTnUY~T~G GAGuY~aG A~D3~PG
841 G3C~5TT GAY~Y3G ID~G~IG GC~3~XP~ G3~YaIG GTTT~A~
901 CGr~r ~ CG~ q~ G~ ~aCAA
i 7 2 .....3
961 AACCTCG~AA ~CAP~a~ CAAA~I~ Gl~l~C:~T G(~CCI~GA ~A
1021 ICP~AA C~C~aG~T TI~A~AA CA~l~AA G~ AAP~A~
1081 AGICGGl~A Gl~ G~aG~AA ~C Al~aGlOE~ ~ACG~
1141 AGI~;CG~A GITll~CCI~ I~CGCP~QI~ 1~1~ ACCICGCC~ TlTICCGl~A
1201 ~A~ C~ ~A~ G~AA~ A~OEA~G~ ACrITl~T
1261 ~1~(3Gl~ ACl~l~ (~ CI~G:G APIOEG~C A~;CCCG~C
1321 A~CGA~ m~;;CG Al~CGCCA~ e~Aa A~A A~I~AC
1381 A~Ga~ ~1~ ~CGl'r ~I~T C~I~G A(i~CA
1441 G~l~aA~ A~A~ C~AAA Gl~ Grl~A AaAACA~A
1501 CG~ A~AAMI~l~ Tl~ Cl~;A A~IT TrlTl~A~
1561 Ah~A Crl~Gl:G Al~l~G~ AU~I~al~ CC~ACGA A~G~C
1621 C~l~A IT:I~CC CAG~I~A A(rl~lTr Iq~l~ AA~TI~A
1681 ~A~ A~ 1~3;~C AOE~G AS ~ IW~I~ AITm~;AI~
1741 AA~Clq~; C~ACAA AGAI~A ~ Al~l~ ACGAll~CA
1801 A~ TI~ C~I~ At~ Tll~l~Cr AMG;a~:; G~AAG~
1861 ~mI~ ~1~;~ 1~GI~3GCGA AGA~ I~AI~C CA~AGa~G
1921 ~mA ~11~ I~A ~s~ G~CGIITCCT CCAGA6rm
1981 G~ACCl~IX3G G~AA ~9a~I~ CA~GCGC CG~C~CGCC GGCGl~A
2041 AGC~ll~IGlT TmGCGGAl'G AG~G~AG~ Tl~l&A ~Ca~TTAA AI~CGC
-
~ 3
2101 ~LaPGCYG~ qGa~A~Z~ GaAITrG~CT GECGaC~GI~ GCGCGETa~T CC ~ C
2161 CCCATGCCGA ALICAEPAEr GAAACGCCGT AGCGCCGAIG GIPGrGnaGG GTCTCCCCAT
2221 GCGAEAGIAG GGAALTGCCA GGCA~CAAAr AAAP~GAAAG GC~X~GTCGA AAG~LqGEGC
2281 cmcGTTTT P~CqGTnarT TGTCGG~G~A CGCTCTCCTG AEn~GGaCAA ATCCGCCGGG
2341 AGCGG~ITTG A~CGTTGCGA ~ CGGCC CGGAGGaTGa CGGGCALGaC GCCCGCCAIA
2401 AaCTGCCAGG CA~CAAArT~ AGCAAaYGC CArGCIGaDG Ga~rGOCm TIscGrTTcT
2461 A~aaAclcsr TTGTTT~m TTCqAAA~AC ATlCaAArEr GTa3CCGrTC A~GaGa~AAr
2521 AaCCCTGaT~ AA5GCTICAA TPP~ATlraA AAAGGaAaaa TATGaGTa~T CPALAT51CC
2581 GTGrCGCCCT TAlTcccm ~ LGGCAr mGccTlcc IGTTTTTGCT CACCCAGAAA
2641 CGCTGGT~aa PGTPAAAGAT GCTGh GA5C h~ L ACG~GTGGGT TPL~IOEaAC
2701 TG34ICTCAA CAGCGG~AG ATCCrr~AEA Gl m CGCCC CGAAGAACGr m CCAE~A
2761 TGAGCAC m TAAE3TIe5G CrATGTGGCG CGGrATTAlC CCGTGil~aC GCCGG~CA~G
2821 AGCAACICGG TCGCCGCArA CACraq~lCrC AG~ADGACrT GGTIGAGTAC TCACCAGTC~
2881 C~GAAAAGCA TcTTAcGGAT GGCA~GACaG TAhGhGAATT ATGCAGTGCT GCC~IAALCA
2941 TGKGIGATAA CACra~GGCC AE~rrYCT~C 'r~AAOGAT CGG~GGACCG AE GaGCTAa
3001 CCLrTI~ m G~ACAACAIG C~ 5~P~G TAE~n3GCCT TGEn~GrIGG GAACCGGAGC
3061 rrGAA3~aAGC CATACCaaAC GACG~GCGTG AC~CCALCaT GCCrx AGcA ATGG AACAA
3121 CG`TTGCGCAA ACTATTAACT GGCGAACI~C TTACrCTaGC TrCCCGGCAA CAErrAAr~G
3181 ACra~AIGGA GGCGGArAAA GTTGCAGG~C CACrr-TGCG CTCGGCCCrr CCGGCTGGCT
s~ ~
3301 ~ ~ I~CCG~CG ~1~ CP~GGGG A~
3361 C~P~ ACG~A~ ~G
3421 M~l~l~a CCaal~ll~ ~T~C qT~G~G~ TI~AaAC
3481 ~AA~ C~I~AG Anrl~ P~a~ GPCC~ CCTI~ACGI~
3541 A~lll~CGl~ CC;Q~;;~G ~G;CCCCG q~G~a~ C~A;i~ ~1~
3601 cmTm~ Gt~GC~IC ~1~1~: AA~A~ A~CGCI~ CCAGCGGI~;
3661 I~lTL~ ~:5~aG C~X:A~
3721 C~CC ~aP3~ Cl~l~ IY~ ~cl~ac ~
3841 ~a~ Al~;CGC ~C
3901 GGDCGGGC5G AACGGGGGGT TCGnGCALaC PGlrCaGCTT GG~GOGaACG ~ CCG
3961 AaL5GAGæ~ CCTPCAGCGT GAGCArTGAG AAAGCGCCAC GC~IOOOGAA GGGAGAApGG
4021 CGGACAG31A TCCGGTAA~C GacAGGGTcG GAACaGC~G~ GCGCALGAGG GAGCTTCCAG
4081 GGGEAAACGC CTGGr~5CTT TP~AGTCCTG ICGGGnT~CG CCACrICTG~ CTTGAGCGTC
4141 GATTTTT35G PIGCTCGTCA GGGGGGCGGA GCCIA5GG~A AAACGCCA~C AACGCGGCCT
4201 TTTIACGGIT CCTGGCCTTT IGCTGGCCTT TT~ITCACAr GTTCTTTCCT GCGTT~ICCC
4261 CTGaTlCTGr GGAI~ACCGT ATTACCGCCT TTGAGnGA~C TGAIACCGCT CGCCGCAGCC
4321 GAACGACCGA GCGCAGCGAG TCAGnGAGCG AGGAAGCGGA AGAGCGCCTG AIGCGGqA~T
~381 II~CCI'r~C ~:A~l~IOE GGI~P~lTI~C ACCOE~I~ GI~CI~IC P~aA~
4441 ~I~1~5~ C(~A~ P~ C~CICCGCI~ TCGCI~CGIG 1~1~
~1501 GGC~CGCCC CGPL~GC CA~CGC l'OEGCGCCC IGI~ Gl~l~CCC
4561 GGCP~CCGCI ~C1~P~ ~l~P~ CICCGGG~C TGC~IGI~C AQ~ll
4621 ACCGI~I~ CCG~CG CGZ~;GC~
SEQ ID NO: 2
TYPE OF SEQUENCE: Amino acid sequence
LENGTH OF SEQUENCE: 770 amino acids
FORM OF STRAND: single strand
~Set Tyr Tyr Leu Glu Phe Gln Gln Gln Gln Gln Leu Leu Asp Val Val 16
Lys Arg Gln Gln Glu Leu Leu Arg Leu Thr Val Trp Gly Thr Lys Asn 32
Leu Gln Ala Arg Val Thr Ala Ile Glu Lys Tyr Leu Gln Asp Gln Ala 48
Arg Leu Asn Ser Trp Gly Cys Ala Phe Arg Gln Val Cys His Thr Thr 64
Val Pro Trp Val Asn Asp Ser Leu Ala Pro Asp Trp Asp Asn Met Thr 80
Trp Gln Glu Trp Glu Lys Gln Val Arg Tyr Leu Glu Ala Asn Ile Ser 96
Lys Ser Leu Glu Gln Ala Gln Ile Gln Gln Glu Lys Asn Met Tyr Glu 112
Leu Gln Lys Leu Asn Ser Trp Asp Asp Pro Leu Glu Ser Cys Asp Lys 128
Cys Gln Leu Lys Gly Glu Ala Met His Gly Gln Val Asp Cys Ser Pro 144
Gly Ile Trp Gln Leu Asp Cys Thr HiS Leu Glu Gly Lys Ile Ile Leu 160
Val Ala Val His Val Ala Ser Gly Tyr Ile Glu Ala Glu Val Ile Pro 176
Ala Glu Thr Gly Gln Glu Thr Ala Tyr Phe Ile Leu Lys Leu Ala Gly 192
Arg Trp Pro Val Lys Val Ile HiS Thr Asp Asn Gly Ser Asn Phe Thr 208
Ser Thr Thr Val Lys Ala Ala Cys Trp Trp Ala Gly Ile Lys Gln Glu 224
Phe Gly Ile Pro Tyr Asn Pro Gln Ser Gln Gly Val Val Glu Ser Met 240
Asn Lys Glu Leu Ly~ Lys Ile Ile Gly Gln Val Arg Asp Gln Ala Glu 256
His Leu Lys Thr Ala Val Gln Met Ala Val Phe Ile HiS Asn Phe Lys 272
Arg Lys Gly Gly Ile Gly Gly Tyr Ser Ala Gly Glu Arg Ile Val Asp 288
Ile Ile Ala Thr Asp Ile Gln Thr Lys Glu Leu Gln Lys Gln Ile Ile 304
Lys Ile Gln Asn Phe Arg Val Tyr Tyr Arg Asp Ser Arg Asp Pro Leu 320
Trp Lys Gly Pro Ala Lys Leu Leu Trp Lys Gly Glu Gly Ala Val Val 336
Ile Gln Asp Asn Ser Glu Ile Lys Val Val Pro Arg Arg Lys Ala Lys 352
Ile Ile Arg Asp Tyr Gly Lys Gln Ser Asp Arg Ser Ser Arg Val Gln 368
Thr Arg Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu 384
Arq Ala Ile Glu Thr Gln Gln HiS Leu Leu Gln Leu Thr Val Trp Gly 400
Ile Lys Gln Leu Gln Ala Arg Val Leu Ala Val Glu Arg Tyr Leu Gln 416
Asp Gln Arg Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys 432
Thr Thr Thr Val Pro Trp Asn Thr Ser Trp Ser Asn Lys Ser Leu Asp 448
Thr Ile Trp HiS Asn Met Thr Trp Met Glu Trp Glu Arg Glu Ile Asp 464
Asn Tyr Thr Ser Ser Asp Lys Gly Asn Ser Ser Gln Val Se~r Gln Asn 480
Tyr Pro Ile Val Gln Asn Leu Gln Gly Gln Met Val l~is Glll Ala Ile 496
Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Ile G1U Glu Lys Ala 512
Phe Ser Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser Glu Gly Ala 528
Thr Pro Gln A~p Leu Asn Thr Met Leu A~n Thr Val Gly G1Y E~is Gln 544
Ala Ala Met Gln Met Leu Lys Glu Thr Ile A~n Glu Glu Ala Ala Glu 560
Trp A~p Arg Val His Pro Val ~-~ic Ala Gly Pro I1Q Ala Pro Gly Gln 576
Met Arg G1U Pro Arg G1Y Ser Asp lle Ala Gly Thr Thr Ser Thr Leu 592
Gln Glu Gln Ile G1Y Trp Met Thr Asn Asn Pro Pro Ile Pro Val Gly 608
Glu Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg 624
Met Tyr Ser Pro Val Ser Ile Leu Asp Ile Arg Gln Gly Pro Lys Glu 640
Yro Phe Arg Asp Tyr Val Asp Arq Phe Tyr Lys Thr Leu Arg Ala G1U 656
(;ln Ala Ser Gln G1U Val Lys Asn Trp Met Thr Glu Thr Leu Leu Val 672
Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Lys Ala Leu Gly Pro 688
Ala Ala Thr Leu Glu G1U Met Met Thr Ala Cys Gln G1Y Val Gly Gly 704
Pro Gly His Lys Ala Arg Val Leu Ala Glu Ala Met Ser Gln Val Thr 720
Asn Ser Ala Thr Ile Met Met Gln Arg Gly Asn Phe Arg Asn Gln Lys 736
Lys Thr Val Lys Cys Phe Asn Cys Gly Lys G1U G1Y His I le Ala Lys 752
Asn Cys Arg Ala Ser Arg Lys Lys Arg Arg Arg Lys Lys Arg Arg Lys 768
Lys Lys 770
