U.S. patent application number 09/996561 was filed with the patent office on 2002-10-17 for process for producing microbial transglutaminase.
This patent application is currently assigned to AJINOMOTO CO., INC.. Invention is credited to Miwa, Tetsuya, Nakamura, Nami, Seguro, Katsuya, Yokoyama, Keiichi.
Application Number | 20020151703 09/996561 |
Document ID | / |
Family ID | 26499683 |
Filed Date | 2002-10-17 |
United States Patent
Application |
20020151703 |
Kind Code |
A1 |
Yokoyama, Keiichi ; et
al. |
October 17, 2002 |
Process for producing microbial transglutaminase
Abstract
Disclosed are a protein having a transglutaminase activity,
which comprises a sequence ranging from serine residue at the
second position to proline residue at the 331st position in an
amino acid sequence represented by SEQ ID No. 1 wherein the
N-terminal amino acid of the protein corresponds to serine residue
at the second position of SEQ ID No. 1, a DNA encoding the protein,
a transformant having the DNA, and a process for producing a
protein having a transglutaminase activity, which comprises the
steps of culturing the transformant in a medium. The protein can be
produced in a large amount with the transformant using a host such
as E. coli.
Inventors: |
Yokoyama, Keiichi;
(Kawasaki-shi, JP) ; Nakamura, Nami;
(Kawasaki-shi, JP) ; Miwa, Tetsuya; (Kawasaki-shi,
JP) ; Seguro, Katsuya; (Kawasaki-shi, JP) |
Correspondence
Address: |
OBLON SPIVAK MCCLELLAND MAIER & NEUSTADT PC
FOURTH FLOOR
1755 JEFFERSON DAVIS HIGHWAY
ARLINGTON
VA
22202
US
|
Assignee: |
AJINOMOTO CO., INC.
15-1, Kyobashi 1-chome, Chuo-ku
Tokyo
JP
|
Family ID: |
26499683 |
Appl. No.: |
09/996561 |
Filed: |
November 30, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09996561 |
Nov 30, 2001 |
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09448310 |
Nov 24, 1999 |
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09448310 |
Nov 24, 1999 |
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09109063 |
Jul 2, 1998 |
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Current U.S.
Class: |
536/23.2 ;
435/252.33; 435/320.1; 435/69.1 |
Current CPC
Class: |
C12N 9/1044
20130101 |
Class at
Publication: |
536/23.2 ;
435/69.1; 435/252.33; 435/320.1 |
International
Class: |
C07H 021/04; C12P
021/02; C12N 001/21 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 4, 1997 |
JP |
180010 |
Claims
What is claimed is:
1. A protein having a transglutaminase activity, which comprises a
sequence ranging from serine residue at the second position to
proline residue at the 331st position in an amino acid sequence
represented by SEQ ID No. 1 wherein the N-terminal amino acid of
the protein corresponds to serine residue at the second position of
SEQ ID No. 1.
2. The protein of claim 1 which consists of an amino acid sequence
of from serine residue at the second position to proline residue at
the 331st position in an amino acid sequence of SEQ ID No. 1.
3. A DNA which encodes for the protein of claim 1.
4. A DNA which encodes for the protein of claim 2.
5. The DNA of claim 3 wherein a base sequence encoding for Arg at
the forth position from the N-terminal amino acid is CGT or CGC,
and a base sequence encoding for Val at the fifth position from the
N-terminal amino acid is GTT or GTA.
6. The DNA of claim 5 wherein a base sequence encoding for from the
N-terminal amino acid to the fifth amino acid, Ser-Asp-Asp-Arg-Val,
has th e following sequence.
5 Ser : TCT or TCC Asp : GAC or GAT Asp : GAC or GAT Arg : CGT or
CGC Val : GTT or GTA
7. The DNA of claim 6 wherein a base sequence encoding for an amino
acid sequence of from the N-terminal amino acid to the fifth amino
acid, Ser-Asp-Asp-Arg-Val, has the sequence
TCT-GAC-GAT-CGT-GTT.
8. The DNA of claim 6 wherein a base sequence encoding for an amino
acid sequence of from the sixth amino acid to the ninth amino acid
from the N-terminal amino acid, Thr-Pro-Pro-Ala, has the following
sequence.
6 Thr : ACT or ACC Pro : CCA or CCG Pro : CCA or CCG Ala : GCT or
GCA
9. The DNA of claim 7 wherein a base sequence encoding for an amino
acid sequence of from the sixth amino acid to the ninth amino acid
from the N-terminal amino acid, Thr-Pro-Pro-Ala, has the following
sequence.
7 Thr : ACT or ACC Pro : CCA or CCG Pro : CCA or CCG Ala : GCT or
GCA
10. A DNA comprising a sequence ranging from thymine base at the
fourth position to guanine base at the 993rd position in the base
sequence of SEQ ID No. 2.
11. A DNA consisting of a sequence ranging from thymine base at the
fourth position to guanine base at the 993rd position in the base
sequence of SEQ ID No. 2.
12. A recombinant DNA having a DNA of claim 3.
13. A recombinant DNA having a DNA of claim 5.
14. A recombinant DNA having a DNA of claim 6.
15. The recombinant DNA of claim 12 which has a promoter selected
from the group consisting of trp, tac, lac, trc, .lambda.PL and
T7.
16. The recombinant DNA of claim 13 which has a promoter selected
from the group consisting of trp, tac, lac, trc, .lambda.PL and
T7.
17. The recombinant DNA of claim 14 which has a promoter selected
from the group consisting of trp, tac, lac, trc, .lambda.PL and
T7.
18. A transformant obtained by the transformation with the
recombinant DNA of claim 12.
19. The transformant of claim 18 wherein a transformation is
conducted by use of a multi-copy vector.
20. The transformant of claim 18, which belongs to Escherichia
coli.
21. A transformant obtained by the transformation with the
recombinant DNA of claim 14 wherein a transformation is conducted
by use of a multi-copy vector, the transformant belonging to
Escherichia coli.
22. A process for producing a protein having a transglutaminase
activity, which comprises the steps of culturing the transformant
of claim 18 in a medium to produce the protein having the
transglutaminase activity and recovering the protein.
23. The process of claim 22 wherein the transformant is that of
claim 19.
24. The process of claim 22 wherein the transformant is that of
claim 20.
25. The process of claim 22 wherein the transformant is that of
claim 21.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to a protein having a
transglutaminase activity, DNA which encodes for the protein, and a
process for producing the protein. In particularly, the present
invention relates to a process for producing a protein having a
transglutaminase activity by a genetic engineering technique.
[0002] Transglutaminase is an enzyme which catalyzes the acyl
transfer reaction of a .gamma.-carboxyamido group in a peptide
chain of a protein. When such an enzyme react with the protein, a
reaction of an .epsilon.-(.gamma.-Glu)-Lys forming reaction or
substitution reaction of Gln with Glu by the deamidation of Glu can
occur.
[0003] The transglutaminase is used for the production of gelled
foods such as jellies, yogurts, cheeses, gelled cosmetics, etc. and
also for improving the quality of meats [see Japanese Patent
Publication for Opposition Purpose (hereinafter referred to as "J.
P. KOKOKU") No. Hei 1-50382]. The transglutaminase is also used for
the production of a material for microcapsules having a high
thermal stability and a carrier for an immobilized enzyme. The
transglutaminase is thus industrially very useful.
[0004] As for transglutaminases, those derived from animals and
those derived from microorganisms (microbial transglutaminase;
hereinafter referred to as "MTG") have been known hitherto.
[0005] The transglutaminases derived from animals are calcium
ion-dependent enzymes which are distributed in organs, skins and
bloods of animals. They are, for example, guinea pig liver
transglutaminase [K.Ikura et al., Biochemistry 27, 2898 (1988)],
human epidermis keratin cell transglutaminase [M. A. Philips et
al., Proc. Natl. Acad. Sci. USA 87, 9333 (1990)] and human blood
coagulation factor XIII (A. Ichinose et al., Biochemistry 25, 6900
(1990)].
[0006] As for the transglutaminases derived from microorganisms,
those independent on calcium were obtained from microorganisms of
the genus Streptoverticillium. They are, for example,
Streptoverticillium griseocarneum IFO 12776, Streptoverticillium
cinnamoneum sub sp. cinnamoneum IFO 12852 and Streptoverticillium
mobaraense IFO 13819 [see Japanese Patent Unexamined Published
Application (hereinafter referred to as "J. P. KOKAI") No. Sho
64-27471].
[0007] According to the peptide mapping and the results of the
analysis of the gene structure, it was found that the primary
structure of the transglutaminase produced by the microorganism is
not homology with that derived from the animals at all (European
Patent publication No. 0 481 504 A1).
[0008] Since the transglutaminases (MTG) derived from
microorganisms are produced by the culture of the above-described
microorganisms followed by the purification, they had problems in
the supply amount, efficiency, and the like. It is also tried to
produce them by a genetic engineering technique. This technique
includes a process which is conducted by the secretion expression
of a microorganism such as E. coli, yeast or the like (J. P. KOKAI
No. Hei 5-199883), and a process wherein MTG is expressed as an
inactive fusion protein inclusion body in E. coli, this inclusion
body is solubilized with a protein denaturant, the denaturant is
removed and then MTG is reactivated to obtain the active MTG (J. P.
KOKAI No. Hei 6-30771).
[0009] However, these processes have problems when they are
practiced on an industrial scale. Namely, when the secretion by the
microorganisms such as E. coli and yeast is employed, the amount of
the product is very small; and when MTG is obtained in the form of
the inactive fusion protein inclusion body in E. coli, an expensive
enzyme is necessitated for the cleavage.
[0010] It is known that when a foreign protein is secreted by the
genetic engineering method, the amount thereof thus obtained is
usually smal 1. On the contrary, it is also known that when the
foreign protein is produced in the cell of E. coli, the product is
in the form of inert protein inclusion body in many cases although
the expressed amount is high. The protein inclusion body must be
solubilized with a denaturant, the denaturating agent must be
removed and then MTG must be reactivated.
[0011] It is already known that in the expression in E. coli, an
N-terminal methionine residue in natural protein obtained after the
translation of gene is efficiently cleaved with methionine
aminopeptidase. However, the N-terminal methionine residue is not
always cleaved in an exogenous protein.
[0012] Processes proposed hitherto for obtaining a protein free
from N-terminal methionine residue include a chemical process
wherein a protein having methionine residue at the N-terminal or a
fusion protein having a peptide added thereto through methionine
residue is produced and then the product is specifically decomposed
at the position of methionine residue with cyanogen bromide; and an
enzymatic process wherein a recognition sequence of a certain
site-specific proteolytic enzyme is inserted between a suitable
peptide and an intended peptide to obtain a fusion peptide, and the
site-specific hydrolysis is conducted with the enzyme.
[0013] However, the former process cannot be employed when the
protein sequence contains a methionine residue, and the intended
protein might be denatured in the course of the reaction. The
latter process cannot be employed when a sequence which is easily
broken down is contained in the protein sequence because the yield
of the intended protein is reduced. In addition, the use of such a
proteolytic enzyme is unsuitable for the production of protein on
an industrial scale from the viewpoint of the cost.
[0014] Conventional processes for producing MTG have many problems
such as supply amount and cost. Namely, in the secretion expression
by E. coli, yeast or the like, the expressed amount is
disadvantageously very small. In the production of the fusion
protein inclusion body in E. coli, it is necessary, for obtaining
mature MTG, to cleave the fusion part with restriction protease
after the expression. Further, it has been found that since MTG is
independent on calcium, the expression of active MTG in the cell of
a microorganism is fatal because this enzyme acts on the
endoprotein.
[0015] Thus, for the utilization of MTG, produced by the gene
recombination, on an industrial scale, it is demanded to increase
the production of mature MTG free of the fusion part. The present
invention has been completed for this purpose. The object of the
present invention is to product MTG in a large amount in
microorganisms such as E. coli.
[0016] When MTG is expressed with recombinant DNA of the present
invention, methionine residue is added to the N-terminal of MTG.
However, by the addition of the methionine residue to the
N-terminal of MTG, there is some possibility wherein problems of
the safety such as impartation of antigenicity to MTG occur. It is
another problem to be solved by the present invention to produce
MTG free of methionine residue corresponding to the initiation
codon.
SUMMARY OF THE INVENTION
[0017] An object of the present invention is to provide a novel
protein having a transglutaminase activity.
[0018] Another object of the present invention is to provide a DNA
encoding for the novel protein having a transglutaminase
activity.
[0019] Another object of the present invention is to provide a
recombinant DNA encoding for the novel protein having a
transglutaminase activity.
[0020] Another object of the present invention is to provide a
transformant obtained by the transformation with the recombinant
DNA.
[0021] Another object of the present invention is to provide a
process for producing a protein having a transglutaminase
activity.
[0022] These and other objects of the present invention will be
apparent from the following description and examples.
[0023] For solving the above-described problems, the inventors have
constructed a massive expression system of protein having
transglutaminase activity by changing the codon to that for E.
coli, or preferably by using a multi-copy vector (pUC19) and a
strong promoter (trp promoter).
[0024] Since MTG is expressed and secreted in the prepro-form from
microorganisms of actinomycetes, the MTG does not have methionine
residue corresponding to the initiation codon at the N-terminal,
but the protein expressed by the above-described expression method
has the methionine residue at the N-terminal thereof. To solve this
problem, the inventors have paid attention to the substrate
specificity of methionine aminopeptidase of E. coli, and succeeded
in obtaining a protein having transglutaminase activity and free
from methionine at the N-terminal by expressing the protein in the
form free from the aspartic acid residue which is the N-terminal
amino acid of MTG. The present invention has been thus
completed.
[0025] Namely, the present invention provides a protein having a
transglutaminase activity, which comprises a sequence ranging from
serine residue at the second position to proline residue at the
331st position in an amino acid sequence represented by SEQ ID No.
1 wherein N-terminal amino acid of the protein corresponds to
serine residue at the second position of SEQ ID No. 1.
[0026] There is provided a protein which consists of an amino acid
sequence of from serine residue at the second position to proline
residue at the 331st position in an amino acid sequence of SEQ ID
No. 1.
[0027] There is provided a DNA which codes for said proteins.
[0028] There is provided a recombinant DNA having said DNA, in
particular, a recombinant DNA expressing said DNA.
[0029] There is provided a transformant obtained by the
transformation with the recombinant DNA.
[0030] There is provided a process for producing a protein having a
transglutaminase activity, which comprises the steps of culturing
the transformant in a medium to produce the protein having a
transglutaminase activity and recovering the protein.
[0031] Taking the substrate specificity of methionine
aminopeptidase into consideration, the process for producing the
protein having transglutaminase activity and free of initial
methionine is not limited to the removal of the N-terminal aspartic
acid.
BRIEF EXPLANATION OF THE DRAWINGS
[0032] FIG. 1 shows a construction scheme of MTG expression plasmid
pTRPMTG-01.
[0033] FIG. 2 shows a construction scheme of MTG expression plasmid
pTRPMTG-02.
[0034] FIG. 3 is an expansion of SDS-polyacrylamide electrophoresis
showing that MTG was expressed.
[0035] FIG. 4 shows a construction scheme of MTG expression plasmid
pTRPMTG-00.
[0036] FIG. 5 shows a construction scheme of plasmid pUCN216D.
[0037] FIG. 6 shows a construction scheme of MTG expression plasmid
pUCTRPMTG(+)D2.
[0038] FIG. 7 shows that GAT corresponding to Aspartic acid residue
is deleted.
[0039] FIG. 8 shows that N-terminal amino acid is serine.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0040] The proteins having a transglutaminase activity according to
the present invention comprise a sequence ranging from serine
residue at the second position to proline residue at the 331st
position in an amino acid sequence represented by SEQ ID No. 1 as
an essential sequence but the protein may further have an amino
acid or amino acids after proline residue at the 331st position.
Among these, the preferred is a protein consisting of an amino acid
sequence of from serine residue at the second position to proline
residue at the 331st position in an amino acid sequence of SEQ ID
No. 1.
[0041] In these amino acid sequences, the present invention
includes amino acid sequences wherein an amino acid or some amino
acids are delete d, substituted or inserted as far as such amino
acid sequences have a transglutaminase activity.
[0042] The DNA of the present invention encodes the above-mentioned
proteins. Among these, the preferred is a DNA wherein a base
sequence encoding for Arg at the forth position from the N-terminal
amino acid is CGT or CGC, and a base sequence encoding for Val at
the fifth position from the N-terminal amino acid is GTT or GTA.
Furthermore, the preferred is a DNA wherein a base sequence
encoding for the N-terminal amino acid to fifth amino acid,
Ser-Asp-Asp-Arg-Val, has the following sequence.
1 Ser : TCT or TCC Asp : GAC or GAT Asp : GAC or GAT Arg : CGT or
CGC Val : GTT or GTA
[0043] In this case, the preferred is a DNA wherein a base sequence
encoding for amino acid sequence of from the N-terminal amino acid
to fifth amino acid, Ser-Asp-Asp-Arg-Val, has the sequence
TCT-GAC-GAT-CGT-GTT.
[0044] Furthermore, the preferred is a DNA wherein a base sequence
10 encoding for amino acid sequence of from sixth amino acid to
ninth amino acid from the N-terminal amino acid, Thr-Pro-Pro-Ala,
has the following sequence.
2 Thr : ACT or ACC Pro : CCA or CCG Pro : CCA or CCG Ala : GCT or
GCA
[0045] Furthermore, the preferred is a DNA comprising a sequence
ranging from thymine base at the fourth position to guanine base at
the 993rd position in the base sequence of SEQ ID No. 2. In this
case, more preferred is a DNA consisting of a sequence ranging from
thymine base at the fourth position to guanine base at the 993rd
position in the base sequence of SEQ ID No. 2.
[0046] In the DNA sequences mentioned above, nucleic acids encoding
an amino acid or some amino acids may be deleted, substituted or
inserted as far as such DNA encodes an amino acid sequence having a
transglutaminase activity.
[0047] The recombinant DNA of the present invention has one of DNA
mentioned above. In this case, the preferred is a DNA having a
promoter selected from the group consisting of trp, tac, lac, trc,
.lambda.PL and T7.
[0048] The transformants of the present invention are obtained by
the transformation with the above-mentioned recombinant DNA. Among
these, it is preferable that a transformation be conducted by use
of a multi-copy vector, and that the transformants belong to
Escherichia coli.
[0049] The process for producing a protein having a
transglutaminase activity according to the present invention
comprises the steps of culturing one of the above-mentioned
transformants in a medium to produce the protein having a
transglutaminase activity and recovering the protein.
[0050] The detailed description will be further made on the present
invention.
[0051] (1) It is known that the expression of MTG in the cells of a
microorganism is fatal. It is also known that in the high
expression of the protein in a microorganism such as E. coli, the
expressed protein is inclined to be in the form of inert insoluble
protein inclusion bodies. Under these circumstances, the inventors
made investigations for the purpose of obtaining a high expression
of MTG as an inert, insoluble protein in E. coli.
[0052] A structural gene of MTG used for achieving the high
expression is a DNA containing a sequence ranging from thymine base
at the fourth position to guanine base at the 993rd position in the
base sequence of SEQ ID No. 2. Taking the degeneration of the
genetic codon, the third letter in the degenerate codon in a domain
which codes for the N-terminal portion is converted to a codon rich
in adenine and uracil and the remaining portion is comprised of a
codon frequently used for E. coli in order to inhibit the formation
of high-order structure of mRNA, though a DNA which codes for
proteins having the same amino acid sequence can have various base
sequences.
[0053] A strong promoter usually used for the production of foreign
proteins is used for the expression of MTG structural gene, and a
terminator is inserted into the downstream of MTG structural gene.
For example, the promoters are trp, tac, lac, trc, .lambda.PL and
T7, and the terminators are trpA, lpp and T4.
[0054] For the efficient translation, the variety and number in the
SD sequence, and the base composition, sequence and length in the
domain between the SD sequence and initiation codon were optimized
for the expression of MTG.
[0055] The domain ranging from the promoter to the terminator
necessitated for the expression of MTG can be produced by a
well-known chemical synthesis method. An example of the base
sequence is shown in SEQ ID No. 3. In the amino acid sequence of
sequence No. 3, aspartic acid residue follows the initiation codon.
However, this aspartic acid residue is preferably removed as will
be described below.
[0056] The present invention also provides a recombinant DNA usable
for the expression of MTG.
[0057] The recombinant DNA can be produced by inserting a DNA
containing the structural gene of the above-described MTG in a
known expression vector selected depending on a desired expression
system. The expression vector used herein is preferably a
multi-copy vector.
[0058] Known expression vectors usable for the production of the
recombinant DNA of the present invention include pUC19 and pHSG299.
An example of the recombinant DNA of the present invention obtained
by integrating DNA of the present invention into pUC19 is
pUCTRPMTG-02(+).
[0059] The present invention also relates to various transformants
obtained by the introduction of the recombinant DNA.
[0060] The cells capable of forming the transformant include E.
coli and the like.
[0061] An example of E. coli is the strain JM109 (recAl, endAl,
gyrA96, thi, hsdR17, supE44, relAl,.DELTA. (lac-proAB)/F' [traD36,
proAB+, laciq, lacZ .DELTA.M15]).
[0062] A protein having a transglutaminase activity is produced by
culturing the transformant such as that obtained by transforming E.
coli JM109 with pUCTRPMTG-02(+) which is a vector of the present
invention.
[0063] Examples of the medium used for the production include 2xYT
medium used in the Example given below and medium usually used for
culturing E. coli such as LB medium and M9-Casamino acid
medium.
[0064] The culture conditions and production-inducing conditions
are suitably selected depending on the kinds of the vector,
promoter, host and the like. For example, for the production of a
recombinant product with trp promoter, a chemical such as
3-.beta.-indoleacrylic acid may be used for efficiently working the
promoter. If necessary, glucose, Casamino acid or the like can be
added in the course of the culture. Further, a chemical
(ampicillin) resistant to genes which are resistant to chemicals
kept in plasmid can also be added in order to selectively
proliferate a recombinant E. coli.
[0065] The protein having a transglutaminase activity, which is
produced by the above-described process, is extracted from the
cultured strain as follows: After the completion of the culture,
the cells are collected and suspended in a buffer solution. After
the treatment with lysozyme, freezing/melting, ultrasonic
disintegration, etc., the thus-obtained suspension of the
disintegrated cells is centrifuged to divide it into a supernatant
liquid and precipitates.
[0066] The protein having a transglutaminase activity is obtained
in the form of a protein inclusion body and contained in the
precipitates. This protein is solubilized with a denaturant or the
like, the denaturant is removed and the protein is separated and
purified. Examples of the denaturants usable for solubilizing the
protein inclusion body produced as described above include urea
(such as 8M) and guanidine hydrochloride (such as 6 M). After
removing the denaturant by the dialysis or the like, the protein
having a transglutaminase activity is regenerated. Solutions used
for the dialysis are a phosphoric acid buffer solution, tris
hydrochloride buffer solution, etc. The denaturant can be removed
not only by the dialysis but also dilution, ultrafiltration or the
like. The regeneration of the activity is expectable by any of
these techniques.
[0067] After the regeneration of the activity, the active protein
can be separated and purified by a suitable combination of
well-known separation and precipitation methods such as salting
out, dialysis, ultrafiltration, gel filtration, SDS-polyacrylamide
electrophoresis, ion exchange chromatography, affinity
chromatography, reversed-phase high-performance liquid
chromatography and isoelectric point electrophoresis.
[0068] (2) The present invention provides a protein having a
transglutaminase activity, which has a sequence ranging from serine
residue at the second position to proline residue at the 331st
position in the amino acid sequence represented in SEQ ID No.
1.
[0069] The N-terminals of MTG produced by the product transformed
with recombinant DNA having a DNA represented in SEQ ID No. 3 was
analyzed to find that most of them contained (formyl)methionine
residue of the initiation codon.
[0070] However, when a gene which encodes for an exogenous protein
is expressed in E. coli, the gene is designed so that the intended
protein is positioned after the methionine residue encoded by ATG
which is the translation initiation signal for the gene. It is
already known that N-terminal methionine residues of a natural
protein obtained by the translation from genes are more efficiently
cut by methionine aminopeptidase. However, the N-terminal
methionine residues are not always cut in the exogenous
protein.
[0071] It is known that the substrate specificity of methionine
aminopeptidase varies depending on the variety of the amino acid
residue positioned next to the methionine residue. When the amino
acid residue positioned next to the methionine residue is alanine
residue, glycine residue, serine residue or the like, the
methionine residue is easily cleaved, and when the former is
aspartic acid, asparagine, lysine, arginine, leucine or the like,
the latter is difficultly cleaved [Nature 326, 315(1987)].
[0072] The N-terminal amino acid residue of MTG is aspartic acid
residue. When a methionine residue derived from the initiation
codon is positioned directly before the aspartic acid residue,
methionine aminopeptidase difficultly acts on the obtained
sequence, and the N-terminal methionine residue is usually not
removed but remains. However, since serine residue is arranged next
to N-terminal aspartic acid in MTG, the sequence can be so designed
that the amino acid residue positioned next to methionine residue
derived from the initiation codon will be serine residue (an amino
acid residue on which methionine aminopeptidase easily acts) by
deleting aspartic acid residue. Thus, a protein having a high
transglutaminase activity, from which the N-terminal methionine
residue has been cleaved, can be efficiently produced.
[0073] The recombinant protein thus obtained is shorter than
natural MTG by one amino acid residue, but the function of this
protein is the same as that of the natural MTG. Namely, MTG
activity is not lost by the lack of one amino acid. Although there
is a possibility that a protein having a transglutaminase activity,
from which the methionine residue has not been cleaved, gains a new
antigenicity, it is generally understood that the sequence
shortened by several residues does not gain a new antigenicity
which natural MTG does not have. Thus, there is no problem of the
safety.
[0074] In fact, a sequence of Met-Ser-Asp-Asp-Arg- . . . was
designed by deleting N-terminal aspartic acid residue from
transglutaminase derived from microorganism (MTG), and this was
produced in E. coli. As a result, methionine residue was
efficiently removed and thereby there was obtained a protein having
a sequence of Ser-Asp-Asp-Arg-. . . . It was confirmed that the
specific activity of the thus-obtained protein is not different
from that of natural MTG.
[0075] A process for producing a protein having a transglutaminase
activity, which has a sequence ranging from serine residue at the
second position to proline residue at the 331st position in the
amino acid sequence represented in SEQ ID No. 1 will be described
below.
[0076] That is, a DNA which encodes for a protein having a
transglutaminase activity and having a sequence ranging from serine
residue at the second position to proline residue at the 331st
position in the amino acid sequence represented in SEQ ID No. 1 is
employed as the MTG structural gene present on recombinant DNA
usable for the expression of MTG. Concretely, a DNA having a
sequence ranging from thymine base at the fourth position to
guanine base at the 993rd position in the base sequence of SEQ ID
No. 2 is employed.
[0077] The N-terminal sequence can be altered by an ordinary DNA
recombination technique, or specific site directional mutagenesis
technique, a technique wherein PCR is used for the whole or partial
length of MTG gene, or a technique wherein the part of the sequence
to be altered is exchanged with a synthetic DNA fragment by a
restriction enzyme treatment.
[0078] The transformant thus transformed with the recombinant DNA
is cultured in a medium to produce a protein having a
transglutaminase activity, and the protein is recovered. The
methods for the preparation of the transformant and for the
production of the protein are the same as those described
above.
[0079] Since the protein thus produced has a sequence of Met-Ser- .
. . from which the methionine residue is easily cleaved with
methionine aminopeptidase, the methionine residue is cleaved in the
cell of E. coli to obtain a protein that starts with serine
residue.
[0080] Although MTG having N-terminal methionine residue is not
present in the nature, the inventors have found that in some of
natural MTG, aspartic acid residue is deleted to have N-terminal
serine. Although a protein having N-terminal methionine residue is
thus different from natural MTG in the sequence, a protein having
N-terminal serine residue is included in the sequences of natural
MTG and, in addition, a protein having such a sequence is actually
present in the nature. Thus, it can be said that such MTG is equal
to natural MTG. Namely, in the production of an enzyme to be used
for foods, such as MTG, in which protein antigenicity is a serious
problem, it is important to produce a protein having
transglutaminase activity and also having a sequence equal to that
of natural MTG, or in other words, to produce a sequence from which
the N-terminal methionine residue was cleaved.
[0081] The following Examples will further illustrate the present
invention, which by no means limit the invention.
EXAMPLE
[0082] Mass Production of MTG in E. coli:
[0083] <1> Construction of MTG Expression Plasmid
pTRPMTG-01:
[0084] MTG gene has been already completely synthesized, taking the
frequency of using codons of E. coli and yeast into consideration
(J. P. KOKAI No. Hei 5-199883). However, the gene sequence thereof
was not optimum for the expression in E. coli. Namely, all of
codons of thirty arginine residues were AGA (minor codons). Under
these conditions, about 200 bases from the N-terminal of MTG gene
were resynthesized to become a sequence optimum for the expression
of E. coli.
[0085] As a promoter for transcripting MTG gene, trp promoter
capable of easily deriving the transcription in a medium lacking
tryptophane was used. Plasmid pTTG2-22 (J. P. KOKAI No. Hei
6-225775) for the high expression of transglutaminase (TG) gene of
Pagrus major was obtained with trp promoter. The sequence in the
upstream of the TG gene of Pagrus major was designed so that a
foreign protein is highly expressed in E. coli.
[0086] In the construction of pTRPMTG-01, the DNA fragment from
ClaI site in the downstream of trp promoter to BglII site in the
downstream of Pagrus major's TG expression plasmid pTTG2-22 (J. P.
KOKAI Hei 6-225775) was replaced with the ClaI/HpaI fragment of the
synthetic DNA gene and the HpaI/BamHI fragment(small) of pGEM15BTG
(J. P. KOKAI Hei 6-30771).
[0087] The ClaI/HpaI fragment of the Synthetic DNA gene has a base
sequence from ClaI site in the downstream of trp promoter of
pTTG2-22 to translation initiation codon, and 216 bases from the
N-terminal of MTG gene. The base sequence in MTG structural gene
was determined with reference to the frequency of using codon in E
coli so as to be optimum for the expression in E. coli. However,
for avoiding the high-order structure of mRNA, the third letter of
the degenerated codon in the domain of encoding the N-terminal part
was converted to a codon rich in adenine and uracil so as to avoid
the arrangement of the same bases as far as possible.
[0088] The ClaI/Hpal fragment of the Synthetic DNA gene was so
designed that it had EcoRI and HindIII sites at the terminal. The
designed gene was divided into blocks each comprising about 40 to
50 bases so that the + chain and the - chain overlapped each other.
Twelve DNA fragments corresponding to each sequence were
synthesized (SEQ ID Nos. 4 to 15). 5' terminal of the synthetic DNA
was phosphatized. Synthetic DNA fragments to be paired therewith
were annealed, and they were connected with each other. After the
acrylamide gel electrophoresis, the DNA fragments of an intended
size was taken out and integrated in EcoRI/HindIII sites of pUC19.
The sequence was confirmed and the correct one was named pUCN216.
From the pUCN216, a ClaI/HpaI fragment (small) was taken out and
used for the construction of pTRPMTG-01.
[0089] <2> Construction of MTG Expression Plasmid
pTRPMTG-02:
[0090] Since E. coli JM109 keeping pTRPMTG-01 did not highly
express MTG, parts (777 bases) other than the N-terminal altered
parts of MTG gene were altered suitably for E. coli. Since it is
difficult to synthesize 777 bases at the same time, the sequence
was determined, taking the frequency of using codons in E. coli
into consideration, and then four blocks (B1, 2, 3 and 4) therefor,
each comprising about 200 bases, were synthesized. Each block was
designed so that it had EcoRI/HindIII sites at the terminal. The
designed gene was divided into blocks of about 40 to 50 bases so
that the + chain and the - chain overlapped each other. Ten DNA
fragments of the same sequence were synthesized for each block, and
thus 40 blocks were synthesized in total (SEQ ID Nos. 16 to 55). 5'
terminal of the synthetic DNA was phosphatized. Synthetic DNA
fragments to be paired therewith were annealed, and they were
connected with each other. After the acrylamide gel
electrophoresis, DNA of an intended size was taken out and
integrated in EcoRI/HindIII sites of pUC19. The base sequence of
each of them was confirmed and the correct ones were named pUCB1,
B2, B3 and B4. As shown in FIG. 2, B1 was connected with B2, and B3
was connected with B4. By replacing a corresponding part of
pTRPMTG-01 therewith, pTRPMTG-02 was constructed. The sequence of
the high expression MTG gene present on pTRPMTG-02 is shown in SEQ
ID No. 3.
[0091] <3> Construction of MTG Expression Plasmid
pUCTRPMTG-02(+), (-):
[0092] Since E. coli JM109 which keeps the pTRPMTG-02 also did not
highly express MTG, the plasmid was multi-copied. EcoO109I fragment
(small) containing trp promoter of pTRPMTG-02 was smoothened and
then integrated into HincII site of pUC19 which is a multi-copy
plasmid. pUCTRPMTG-02(+) in which lacZ promoter and trp promoter
were in the same direction, and pUCTRPMTG-02(-) in which they were
in the opposite direction to each other were constructed.
[0093] <4>Expression of MTG:
[0094] E. coli JM109 transformed with pUCTRPMTG-02(+) and pUC19 was
cultured by shaking in 3 ml of 2xYT medium containing 150 .mu.g/ml
of ampicillin at 37.degree. C. for ten hours (pre-culture). 0.5 ml
of the culture suspension was added to 50 ml of 2xYT medium
containing 150 .mu.g/ml of ampicillin, and the shaking culture was
conducted at 37.degree. C. for 20 hours.
[0095] The cells were collected from the culture suspension and
broken by ultrasonic disintegration. The results of
SDS-polyacrylamide electrophoresis of the whole fraction, and
supernatant and precipitation fractions both obtained by the
centrifugation are shown in FIG. 3. The high expression of the
protein having a molecular weight equal to that of MTG was
recognized in the whole fraction of broken pUCTRPMTG-02(+)/JM109
cells and the precipitate fraction obtained by the centrifugation.
It was confirmed by the western blotting that the protein was
reactive with mice anti-MTG antibody. The expression of the protein
was 500 to 600 mg/L. A sufficient, high expression was obtained
even when 3-.beta.-indole acrylic acid was not added to the
production medium.
[0096] Further, the western blotting was conducted with MTG
antibody against mouse to find that MTG was expressed only slightly
in the supernatant fraction obtained by the centrifugation and that
the expressed MTG was substantially all in the form of insoluble
protein inclusion bodies.
[0097] <5> Construction of MTG Expression Plasmid
pTRPMTG-00:
[0098] To prove that the change in codon of MTG gene caused a
remarkable increase in the expression, pTRPMTG-00 corresponding to
pTRPMTG-02 but in which MTG gene was changed to a gene sequence
completely synthesized before (J. P. KOKAI No. Hei 6-30771) was
constructed.
[0099] pTRPMTG-00 was constructed by connecting PvuII/PstI fragment
(small) from Pagrus major's TG expression plasmid pTRPMTG-02 with
PstI/HimdIII fragment (small, including PvuII site) and
PvuII/HindIII fragment (small) of pGEM15BTG (J. P. KOKAI No. Hei
6-30771).
[0100] <6> Construction of MTG Expression Plasmid
pUCTRPMTG-00(+), (-):
[0101] pTRPMTG-00 was multi-copied. EcoO109I fragment (small)
containing trp promoter and trpA terminator of pTRPMTG-00 was
smoothened and then integrated into HincII site of pUC19 which is a
multi-copy plasmid. pUCTRPMTG-00(+) in which lacZ promoter and trp
promoter were in the same direction, and pUCTRPMTG-00(-) in which
they were in the opposite direction to each other were
constructed.
[0102] <7> Comparison of MTG Expressions:
[0103] E. coli JM109 transformed with pUCTRPMTG-02 (+) or (-),
PUCTRPMTG-00 (+) or (-), pTRPMTG-02, pTRPMTG-01, PTRPMTG-00 or
pUCl9 was cultured by shaking in 3 ml of 2xYT medium containing 150
.mu.g/ml of ampicillin at 37.degree. C. for ten hours
(pre-culture). 0.5 ml of the culture suspension was added to 50 ml
of 2xYT medium containing 150 .mu.g/ml of ampicillin, and the
shaking culture was conducted at 37.degree. C. for 20 hours.
[0104] The cells were collected from the culture suspension, and
MTG expression thereof was determined to obtain the results shown
in Table 1. It was found that the newly constructed E. coli
containing pTRPMTG-00, PUCTRPMTG-00 (+) or (-) did not highly
express MTG. This result indicate that it is necessary for the high
expression of MTG to change the codon of MTG gene into a codon for
E. coli and also to multi-copy the plasmid.
3 TABLE 1 Strain MTG expression pUCTRPMTG-02(+)/JM109 +++
pUCTRPMTG-02(-)/JM109 +++ pUCTRPMTG-00(+)/JM109 +
pUCTRPMTG-00(-)/JM109 + pTRPMTG-02/JM109 + pTRPMTG-01/JM109 +
pTRPMTG-00/JM109 - pUC19/JM109 - +++: at least 300 mg/l +: 5 mg/l
or below -: no expression
[0105] <8> Analysis of N-terminal Amino Acid of Expressed
MTG:
[0106] The N-terminal amino acid residue of the protein inclusion
bodies of expressed MTG was analyzed to find that about 60% of the
sequence of N-terminal was methionine residue and about 40% thereof
was formylmethionine residue. (Formyl)methionine residue
corresponding to the initiation codon was removed by a technical
idea described below.
[0107] <9> Deletion of N-terminal Aspartic Acid Residue of
MTG:
[0108] A base sequence corresponding to aspartic acid residue (the
N-terminal of MTG) was deleted by PCR using pUCN216 containing 216
bases as the template. pUCN216 is a plasmid obtained by cloning
about 216 bp's containing ClaI-HpaI fragment of N-terminal of MTG
in EcoRI/HindIII site of pUC19. pF01 (SEQ ID No. 56) and pR01 (SEQ
ID No. 57) are primers each having a sequence in the vector. PDELD
(SEQ ID No. 58) is that obtained by deleting a base sequence
corresponding to Asp residue. pHd01 (SEQ ID No. 59) is that
obtained by replacing C with G not to include HindIII site. pF01
and PDELD are sense primers and pR01 and pHd01 are antisense
primers.
[0109] 35 cycles of PCR of a combination of pF01 and pHd01, and a
combination of pELD and pR01 for pUCN216 was conducted at
94.degree. C. for 30 seconds, at 55.degree. C. for one minute and
at 72.degree. C. for two minutes. Each PCR product was extracted
with phenol/chloroform, precipitated with ethanol and dissolved in
100 .mu.l of H.sub.2O.
[0110] 1 .mu.l of each of the PCR products was taken, and they were
mixed together. After the heat denaturation at 94.degree. C. for 10
minutes, 35 cycles of PCR of a combination of pF01 and pHd01 was
conducted at 94.degree. C. for 30 seconds, at 55.degree. C. for one
minute and at 72.degree. C. for two minutes.
[0111] The second PCR product was extracted with phenol/chloroform,
precipitated with ethanol, and treated with HindIII and EcoRI.
After pUC19 subcloning, pUCN216D was obtained (FIG. 5). The
sequence of the obtained pUCN216D was confirmed to be the intended
one.
[0112] <10>Construction of the Plasmid Encoding for MTG which
Lacks N-terminal Aspartic Acid:
[0113] EcoO109I/Hpal fragment (small) of pUCN216D was combined with
Eco0109I/Hpal fragment (large) of pUCBl-1 (plasmid obtained by
cloning HpaII/BglII fragment of MTG gene in EcoRI/HindIII site of
pUC19) to obtain pUCNB1-2D. Further, ClaI/BglII fragment (small) of
pUCNB1-2D was combined with ClaI/B/BglIII fragment (large) of
pUCTRPMTG-02(+) which is a plasmid of high MTG expression to obtain
pUC TRPMTG(+)D2, the expression plasmid of MTG which lacks
N-termianl aspertic acid (FIG. 6). As a result, a plasmid
containing MTG gene lacking GAI corresponding to aspartic acid
residue as shown in FIG. 7 was obtained.
[0114] <11>Expression of the Plasmid Encoding for MTG which
Lacks N-terminal Aspartic Acid:
[0115] E. coli JM109 transformed with pUCTRPMTG(+)D2 was cultured
by shaking in 3 ml of 2xYT medium containing 150 .mu.g/ml of
ampicillin at 37.degree. C. for ten hours (pre-culture). 0.5 ml of
the culture suspension was added to 50 ml of 2xYT medium containing
150 .mu.g/ml of ampicillin, and the shaking culture was conducted
at 37.degree. C. for 20 hours. The cells were broken by the
ultrasonic disintegration. The results of the dyeing with Coomassie
Brilliant Blue dyeing and Western blotting with mouse antiMTG
antibody of the thus obtained supernatant liquid and precipitate
indicated that MTG protein lacking N-terminal aspartic acid residue
was detected in the precipitate obtained by the ultrasonic
disintegration, namely in the insoluble fraction. This fact
suggests that MTG protein lacking N-terminal aspartic acid residue
was accumulated as protein inclusion bodies in the cells.
[0116] The N-terminal amino acid sequence of the protein inclusion
bodies was analyzed to find that about 90% thereof was serine as
shown in FIG. 8.
[0117] The results of the analysis of N-terminal amino acids of
expressed MTG obtained in <8> and <11> were compared
with each other as shown in Table 2. It was found that by deleting
the N-terminal aspartic acid residue from MTG, the initiation
methionine added to the N-terminal of the expressed MTG was
efficiently removed.
4 TABLE 2 N-terminal amino acid Strain f-Met Met Asp Ser
pUCTRPMTG-02(+)/JM109 40% 60% N.D. pUCTRPMTG(+)D2/JM109 N.D. 10% --
90%
[0118] <12> Solubilization of MTG Inclusion Bodies Lacking
N-terminal Aspartic Acid Residue, Renaturation of Activity and
Determination of Specific Activity:
[0119] MTG inclusion bodies lacking aspartic acid was partially
purified by repeating the centrifugation several times, and then
dissolved in 8 M urea [50 mM phosphate buffer (pH 5.5)] to obtain
the 2 mg/ml solution. Precipitates were removed from the solution
by the centrifugation and the solution was diluted to a
concentration of 0.5 M urea with 50 mM phosphate buffer (pH 5.5).
The diluted solution was further dialyzed with 50 mM phosphate
buffer (pH 5.5) to remove urea. According to Mono S column test,
the peak having TG activity was eluted when NaCl concentration was
in the range of 100 to 150 mM. The specific activity of the
fraction was determined by the hydroxamate method to find that the
specific activity of the aspartic acid residue-lacking MTG was
about 30 U/mg. This is equal to the specific activity of natural
MTG. It is thus apparent that the lack of aspartic acid residue
exerts no influence on the specific activity.
Sequence CWU 1
1
62 1 331 PRT Artificial Sequence Description of Artificial
SequenceTRANSGLUTAMINASE 1 Asp Ser Asp Asp Arg Val Thr Pro Pro Ala
Glu Pro Leu Asp Arg Met 1 5 10 15 Pro Asp Pro Tyr Arg Pro Ser Tyr
Gly Arg Ala Glu Thr Val Val Asn 20 25 30 Asn Tyr Ile Arg Lys Trp
Gln Gln Val Tyr Ser His Arg Asp Gly Arg 35 40 45 Lys Gln Gln Met
Thr Glu Glu Gln Arg Glu Trp Leu Ser Tyr Gly Cys 50 55 60 Val Gly
Val Thr Trp Val Asn Ser Gly Gln Tyr Pro Thr Asn Arg Leu 65 70 75 80
Ala Phe Ala Ser Phe Asp Glu Asp Arg Phe Lys Asn Glu Leu Lys Asn 85
90 95 Gly Arg Pro Arg Ser Gly Glu Thr Arg Ala Glu Phe Glu Gly Arg
Val 100 105 110 Ala Lys Glu Ser Phe Asp Glu Glu Lys Gly Phe Gln Arg
Ala Arg Glu 115 120 125 Val Ala Ser Val Met Asn Arg Ala Leu Glu Asn
Ala His Asp Glu Ser 130 135 140 Ala Tyr Leu Asp Asn Leu Lys Lys Glu
Leu Ala Asn Gly Asn Asp Ala 145 150 155 160 Leu Arg Asn Glu Asp Ala
Arg Ser Pro Phe Tyr Ser Ala Leu Arg Asn 165 170 175 Thr Pro Ser Phe
Lys Glu Arg Asn Gly Gly Asn His Asp Pro Ser Arg 180 185 190 Met Lys
Ala Val Ile Tyr Ser Lys His Phe Trp Ser Gly Gln Asp Arg 195 200 205
Ser Ser Ser Ala Asp Lys Arg Lys Tyr Gly Asp Pro Asp Ala Phe Arg 210
215 220 Pro Ala Pro Gly Thr Gly Leu Val Asp Met Ser Arg Asp Arg Asn
Ile 225 230 235 240 Pro Arg Ser Pro Thr Ser Pro Gly Glu Gly Phe Val
Asn Phe Asp Tyr 245 250 255 Gly Trp Phe Gly Ala Gln Thr Glu Ala Asp
Ala Asp Lys Thr Val Trp 260 265 270 Thr His Gly Asn His Tyr His Ala
Pro Asn Gly Ser Leu Gly Ala Met 275 280 285 His Val Tyr Glu Ser Lys
Phe Arg Asn Trp Ser Glu Gly Tyr Ser Asp 290 295 300 Phe Asp Arg Gly
Ala Tyr Val Ile Thr Phe Ile Pro Lys Ser Trp Asn 305 310 315 320 Thr
Ala Pro Asp Lys Val Lys Gln Gly Trp Pro 325 330 2 993 DNA
Artificial Sequence Description of Artificial Sequence SYNTHETIC
DNA 2 gat tct gac gat cgt gtt act cca cca gct gaa cca ctg gat cgt
atg 48 Asp Ser Asp Asp Arg Val Thr Pro Pro Ala Glu Pro Leu Asp Arg
Met 1 5 10 15 cca gat cca tat cgt cca tct tat ggt cgt gct gaa act
gtt gtt aat 96 Pro Asp Pro Tyr Arg Pro Ser Tyr Gly Arg Ala Glu Thr
Val Val Asn 20 25 30 aat tat att cgt aaa tgg caa caa gtt tat tct
cat cgt gat ggt cgt 144 Asn Tyr Ile Arg Lys Trp Gln Gln Val Tyr Ser
His Arg Asp Gly Arg 35 40 45 aaa caa caa atg act gaa gaa caa cgt
gaa tgg ctg tct tat ggt tgc 192 Lys Gln Gln Met Thr Glu Glu Gln Arg
Glu Trp Leu Ser Tyr Gly Cys 50 55 60 gtt ggt gtt act tgg gtt aac
tct ggt cag tat ccg act aac cgt ctg 240 Val Gly Val Thr Trp Val Asn
Ser Gly Gln Tyr Pro Thr Asn Arg Leu 65 70 75 80 gca ttc gct tcc ttc
gat gaa gat cgt ttc aag aac gaa ctg aag aac 288 Ala Phe Ala Ser Phe
Asp Glu Asp Arg Phe Lys Asn Glu Leu Lys Asn 85 90 95 ggt cgt ccg
cgt tct ggt gaa act cgt gct gaa ttc gaa ggt cgt gtt 336 Gly Arg Pro
Arg Ser Gly Glu Thr Arg Ala Glu Phe Glu Gly Arg Val 100 105 110 gct
aag gaa tcc ttc gat gaa gag aaa ggc ttc cag cgt gct cgt gaa 384 Ala
Lys Glu Ser Phe Asp Glu Glu Lys Gly Phe Gln Arg Ala Arg Glu 115 120
125 gtt gct tct gtt atg aac cgt gct cta gag aac gct cat gat gaa tct
432 Val Ala Ser Val Met Asn Arg Ala Leu Glu Asn Ala His Asp Glu Ser
130 135 140 gct tac ctg gat aac ctg aag aag gaa ctg gct aac ggt aac
gat gct 480 Ala Tyr Leu Asp Asn Leu Lys Lys Glu Leu Ala Asn Gly Asn
Asp Ala 145 150 155 160 ctg cgt aac gaa gat gct cgt tct ccg ttc tac
tct gct ctg cgt aac 528 Leu Arg Asn Glu Asp Ala Arg Ser Pro Phe Tyr
Ser Ala Leu Arg Asn 165 170 175 act ccg tcc ttc aaa gaa cgt aac ggt
ggt aac cat gat ccg tct cgt 576 Thr Pro Ser Phe Lys Glu Arg Asn Gly
Gly Asn His Asp Pro Ser Arg 180 185 190 atg aaa gct gtt atc tac tct
aaa cat ttc tgg tct ggt cag gat aga 624 Met Lys Ala Val Ile Tyr Ser
Lys His Phe Trp Ser Gly Gln Asp Arg 195 200 205 tct tct tct gct gat
aaa cgt aaa tac ggt gat ccg gat gca ttc cgt 672 Ser Ser Ser Ala Asp
Lys Arg Lys Tyr Gly Asp Pro Asp Ala Phe Arg 210 215 220 ccg gct ccg
ggt act ggt ctg gta gac atg tct cgt gat cgt aac atc 720 Pro Ala Pro
Gly Thr Gly Leu Val Asp Met Ser Arg Asp Arg Asn Ile 225 230 235 240
ccg cgt tct ccg act tct ccg ggt gaa ggc ttc gtt aac ttc gat tac 768
Pro Arg Ser Pro Thr Ser Pro Gly Glu Gly Phe Val Asn Phe Asp Tyr 245
250 255 ggt tgg ttc ggt gct cag act gaa gct gat gct gat aag act gta
tgg 816 Gly Trp Phe Gly Ala Gln Thr Glu Ala Asp Ala Asp Lys Thr Val
Trp 260 265 270 acc cat ggt aac cat tac cat gct ccg aac ggt tct ctg
ggt gct atg 864 Thr His Gly Asn His Tyr His Ala Pro Asn Gly Ser Leu
Gly Ala Met 275 280 285 cat gta tac gaa tct aaa ttc cgt aac tgg tct
gaa ggt tac tct gac 912 His Val Tyr Glu Ser Lys Phe Arg Asn Trp Ser
Glu Gly Tyr Ser Asp 290 295 300 ttc gat cgt ggt gct tac gtt atc acc
ttc att ccg aaa tct tgg aac 960 Phe Asp Arg Gly Ala Tyr Val Ile Thr
Phe Ile Pro Lys Ser Trp Asn 305 310 315 320 act gct ccg gac aaa gtt
aaa cag ggt tgg ccg 993 Thr Ala Pro Asp Lys Val Lys Gln Gly Trp Pro
325 330 3 1519 DNA Artificial Sequence Description of Artificial
Sequence SYNTHETIC DNA 3 ttcccctgtt gacaattaat catcgaacta
gttaactagt acgcaagttc acgtaaaaag 60 ggtatcgatt agtaaggagg tttaaa
atg gat tct gac gat cgt gtt act cca 113 Met Asp Ser Asp Asp Arg Val
Thr Pro 1 5 cca gct gaa cca ctg gat cgt atg cca gat cca tat cgt cca
tct tat 161 Pro Ala Glu Pro Leu Asp Arg Met Pro Asp Pro Tyr Arg Pro
Ser Tyr 10 15 20 25 ggt cgt gct gaa act gtt gtt aat aat tat att cgt
aaa tgg caa caa 209 Gly Arg Ala Glu Thr Val Val Asn Asn Tyr Ile Arg
Lys Trp Gln Gln 30 35 40 gtt tat tct cat cgt gat ggt cgt aaa caa
caa atg act gaa gaa caa 257 Val Tyr Ser His Arg Asp Gly Arg Lys Gln
Gln Met Thr Glu Glu Gln 45 50 55 cgt gaa tgg ctg tct tat ggt tgc
gtt ggt gtt act tgg gtt aac tct 305 Arg Glu Trp Leu Ser Tyr Gly Cys
Val Gly Val Thr Trp Val Asn Ser 60 65 70 ggt cag tat ccg act aac
cgt ctg gca ttc gct tcc ttc gat gaa gat 353 Gly Gln Tyr Pro Thr Asn
Arg Leu Ala Phe Ala Ser Phe Asp Glu Asp 75 80 85 cgt ttc aag aac
gaa ctg aag aac ggt cgt ccg cgt tct ggt gaa act 401 Arg Phe Lys Asn
Glu Leu Lys Asn Gly Arg Pro Arg Ser Gly Glu Thr 90 95 100 105 cgt
gct gaa ttc gaa ggt cgt gtt gct aag gaa tcc ttc gat gaa gag 449 Arg
Ala Glu Phe Glu Gly Arg Val Ala Lys Glu Ser Phe Asp Glu Glu 110 115
120 aaa ggc ttc cag cgt gct cgt gaa gtt gct tct gtt atg aac cgt gct
497 Lys Gly Phe Gln Arg Ala Arg Glu Val Ala Ser Val Met Asn Arg Ala
125 130 135 cta gag aac gct cat gat gaa tct gct tac ctg gat aac ctg
aag aag 545 Leu Glu Asn Ala His Asp Glu Ser Ala Tyr Leu Asp Asn Leu
Lys Lys 140 145 150 gaa ctg gct aac ggt aac gat gct ctg cgt aac gaa
gat gct cgt tct 593 Glu Leu Ala Asn Gly Asn Asp Ala Leu Arg Asn Glu
Asp Ala Arg Ser 155 160 165 ccg ttc tac tct gct ctg cgt aac act ccg
tcc ttc aaa gaa cgt aac 641 Pro Phe Tyr Ser Ala Leu Arg Asn Thr Pro
Ser Phe Lys Glu Arg Asn 170 175 180 185 ggt ggt aac cat gat ccg tct
cgt atg aaa gct gtt atc tac tct aaa 689 Gly Gly Asn His Asp Pro Ser
Arg Met Lys Ala Val Ile Tyr Ser Lys 190 195 200 cat ttc tgg tct ggt
cag gat aga tct tct tct gct gat aaa cgt aaa 737 His Phe Trp Ser Gly
Gln Asp Arg Ser Ser Ser Ala Asp Lys Arg Lys 205 210 215 tac ggt gat
ccg gat gca ttc cgt ccg gct ccg ggt act ggt ctg gta 785 Tyr Gly Asp
Pro Asp Ala Phe Arg Pro Ala Pro Gly Thr Gly Leu Val 220 225 230 gac
atg tct cgt gat cgt aac atc ccg cgt tct ccg act tct ccg ggt 833 Asp
Met Ser Arg Asp Arg Asn Ile Pro Arg Ser Pro Thr Ser Pro Gly 235 240
245 gaa ggc ttc gtt aac ttc gat tac ggt tgg ttc ggt gct cag act gaa
881 Glu Gly Phe Val Asn Phe Asp Tyr Gly Trp Phe Gly Ala Gln Thr Glu
250 255 260 265 gct gat gct gat aag act gta tgg acc cat ggt aac cat
tac cat gct 929 Ala Asp Ala Asp Lys Thr Val Trp Thr His Gly Asn His
Tyr His Ala 270 275 280 ccg aac ggt tct ctg ggt gct atg cat gta tac
gaa tct aaa ttc cgt 977 Pro Asn Gly Ser Leu Gly Ala Met His Val Tyr
Glu Ser Lys Phe Arg 285 290 295 aac tgg tct gaa ggt tac tct gac ttc
gat cgt ggt gct tac gtt atc 1025 Asn Trp Ser Glu Gly Tyr Ser Asp
Phe Asp Arg Gly Ala Tyr Val Ile 300 305 310 acc ttc att ccg aaa tct
tgg aac act gct ccg gac aaa gtt aaa cag 1073 Thr Phe Ile Pro Lys
Ser Trp Asn Thr Ala Pro Asp Lys Val Lys Gln 315 320 325 ggt tgg ccg
taatgaaagc ttggatctct aattactgga cttcacacag 1122 Gly Trp Pro 330
actaaaatag acatatctta tattatgtga ttttgtgaca tttcctagat gtgaggtgga
1182 ggtgatgtat aaggtagatg atgatcctct acgccggacg catcgtggcc
ggcatcaccg 1242 gcgccacagg tgcggttgct ggcgcctata tcgccgacat
caccgatggg gaagatcggg 1302 ctcgccactt cgggctcatg agcgcttgtt
tcggcgtggg tatggtggca ggccccgtgg 1362 ccgggggact gttgggcgcc
atctccttgc atgcaccatt ccttgcggcg gcggtgctca 1422 acggcctcaa
cctactactg ggctgcttcc taatgcagga gtcgcataag ggagagcgtc 1482
gagagcccgc ctaatgagcg ggcttttttt tcagctg 1519 4 39 DNA Artificial
Sequence Description of Artificial Sequence SYNTHETIC DNA 4
aattcatcga ttagtaagga ggtttaaaat ggattctga 39 5 41 DNA Artificial
Sequence Description of Artificial Sequence SYNTHETIC DNA 5
cgatcgtcag aatccatttt aaacctcctt actaatcgat g 41 6 41 DNA
Artificial Sequence Description of Artificial Sequence SYNTHETIC
DNA 6 cgatcgtgtt actccaccag ctgaaccact ggatcgtatg c 41 7 41 DNA
Artificial Sequence Description of Artificial Sequence SYNTHETIC
DNA 7 gatctggcat acgatccagt ggttcagctg gtggagtaac a 41 8 41 DNA
Artificial Sequence Description of Artificial Sequence SYNTHETIC
DNA 8 cagatccata tcgtccatct tatggtcgtg ctgaaactgt t 41 9 41 DNA
Artificial Sequence Description of Artificial Sequence SYNTHETIC
DNA 9 attaacaaca gtttcagcac gaccataaga tggacgatat g 41 10 41 DNA
Artificial Sequence Description of Artificial Sequence SYNTHETIC
DNA 10 gttaataatt atattcgtaa atggcaacaa gtttattctc a 41 11 41 DNA
Artificial Sequence Description of Artificial Sequence SYNTHETIC
DNA 11 tcacgatgag aataaacttg ttgccattta cgaatataat t 41 12 41 DNA
Artificial Sequence Description of Artificial Sequence SYNTHETIC
DNA 12 tcgtgatggt cgtaaacaac aaatgactga agaacaacgt g 41 13 41 DNA
Artificial Sequence Description of Artificial Sequence SYNTHETIC
DNA 13 gccattcacg ttgttcttca gtcatttgtt gtttacgacc a 41 14 42 DNA
Artificial Sequence Description of Artificial Sequence SYNTHETIC
DNA 14 aatggctgtc ttatggttgc gttggtgtta cttgggttaa ca 42 15 40 DNA
Artificial Sequence Description of Artificial Sequence SYNTHETIC
DNA 15 agcttgttaa cccaagtaac accaacgcaa ccataagaca 40 16 38 DNA
Artificial Sequence Description of Artificial Sequence SYNTHETIC
DNA 16 aattcgttaa ctctggtcag tatccgacta accgtctg 38 17 41 DNA
Artificial Sequence Description of Artificial Sequence SYNTHETIC
DNA 17 cgaatgccag acggttagtc ggatactgac cagagttaac g 41 18 49 DNA
Artificial Sequence Description of Artificial Sequence SYNTHETIC
DNA 18 gcattcgctt ccttcgatga agatcgtttc aagaacgaac tgaagaacg 49 19
49 DNA Artificial Sequence Description of Artificial Sequence
SYNTHETIC DNA 19 ggacgaccgt tcttcagttc gttcttgaaa cgatcttcat
cgaaggaag 49 20 35 DNA Artificial Sequence Description of
Artificial Sequence SYNTHETIC DNA 20 gtcgtccgcg ttctggtgaa
actcgtgctg aattc 35 21 35 DNA Artificial Sequence Description of
Artificial Sequence SYNTHETIC DNA 21 gaccttcgaa ttcagcacga
gtttcaccag aacgc 35 22 48 DNA Artificial Sequence Description of
Artificial Sequence SYNTHETIC DNA 22 gaaggtcgtg ttgctaagga
atccttcgat gaagagaaag gcttccag 48 23 48 DNA Artificial Sequence
Description of Artificial Sequence SYNTHETIC DNA 23 gagcacgctg
gaagcctttc tcttcatcga aggattcctt agcaacac 48 24 42 DNA Artificial
Sequence Description of Artificial Sequence SYNTHETIC DNA 24
cgtgctcgtg aagttgcttc tgttatgaac cgtgctctag aa 42 25 39 DNA
Artificial Sequence Description of Artificial Sequence SYNTHETIC
DNA 25 agctttctag agcacggttc ataacagaag caacttcac 39 26 45 DNA
Artificial Sequence Description of Artificial Sequence SYNTHETIC
DNA 26 aattctctag agaacgctca tgatgaatct gcttacctgg ataac 45 27 50
DNA Artificial Sequence Description of Artificial Sequence
SYNTHETIC DNA 27 cttcttcagg ttatccaggt aagcagattc atcatgagcg
ttctctagag 50 28 49 DNA Artificial Sequence Description of
Artificial Sequence SYNTHETIC DNA 28 ctgaagaagg aactggctaa
cggtaacgat gctctgcgta acgaagatg 49 29 49 DNA Artificial Sequence
Description of Artificial Sequence SYNTHETIC DNA 29 gagaacgagc
atcttcgtta cgcagagcat cgttaccgtt agccagttc 49 30 40 DNA Artificial
Sequence Description of Artificial Sequence SYNTHETIC DNA 30
ctcgttctcc gttctactct gctctgcgta acactccgtc 40 31 39 DNA Artificial
Sequence Description of Artificial Sequence SYNTHETIC DNA 31
ctttgaagga cggagtgtta cgcagagcag agtagaacg 39 32 47 DNA Artificial
Sequence Description of Artificial Sequence SYNTHETIC DNA 32
cttcaaagaa cgtaacggtg gtaaccatga tccgtctcgt atgaaag 47 33 47 DNA
Artificial Sequence Description of Artificial Sequence SYNTHETIC
DNA 33 gataacagct ttcatacgag acggatcatg gttaccaccg ttacgtt 47 34 45
DNA Artificial Sequence Description of Artificial Sequence
SYNTHETIC DNA 34 ctgttatcta ctctaaacat ttctggtctg gtcaggatag atcta
45 35 41 DNA Artificial Sequence Description of Artificial Sequence
SYNTHETIC DNA 35 agcttagatc tatcctgacc agaccagaaa tgtttagagt a 41
36 42 DNA Artificial Sequence Description of Artificial Sequence
SYNTHETIC DNA 36 aattcagatc ttcttctgct gataaacgta aatacggtga tc 42
37 44 DNA Artificial Sequence Description of Artificial Sequence
SYNTHETIC DNA 37 catccggatc accgtattta cgtttatcag cagaagaaga tctg
44 38 48 DNA Artificial Sequence Description of Artificial Sequence
SYNTHETIC DNA 38 cggatgcatt ccgtccggct ccgggtactg gtctggtaga
catgtctc 48 39 48 DNA Artificial Sequence Description of Artificial
Sequence SYNTHETIC DNA 39 gatcacgaga catgtctacc agaccagtac
ccggagccgg acggaatg 48 40 35 DNA Artificial Sequence Description of
Artificial Sequence SYNTHETIC DNA 40 gtgatcgtaa catcccgcgt
tctccgactt ctccg 35 41 36 DNA Artificial Sequence Description of
Artificial Sequence SYNTHETIC DNA 41 cttcacccgg agaagtcgga
gaacgcggga tgttac 36 42 40 DNA Artificial Sequence Description of
Artificial Sequence SYNTHETIC DNA 42 ggtgaaggct tcgttaactt
cgattacggt tggttcggtg 40 43 40 DNA Artificial Sequence Description
of Artificial Sequence SYNTHETIC DNA 43 gtctgagcac cgaaccaacc
gtaatcgaag ttaacgaagc 40 44 44 DNA Artificial Sequence Description
of Artificial Sequence SYNTHETIC DNA 44 ctcagactga agctgatgct
gataagactg tatggaccca tgga
44 45 41 DNA Artificial Sequence Description of Artificial Sequence
SYNTHETIC DNA 45 agcttccatg ggtccataca gtcttatcag catcagcttc a 41
46 39 DNA Artificial Sequence Description of Artificial Sequence
SYNTHETIC DNA 46 aattcccatg gtaaccatta ccatgctccg aacggttct 39 47
42 DNA Artificial Sequence Description of Artificial Sequence
SYNTHETIC DNA 47 cacccagaga accgttcgga gcatggtaat ggttaccatg gg 42
48 41 DNA Artificial Sequence Description of Artificial Sequence
SYNTHETIC DNA 48 ctgggtgcta tgcatgtata cgaatctaaa ttccgtaact g 41
49 42 DNA Artificial Sequence Description of Artificial Sequence
SYNTHETIC DNA 49 cttcagacca gttacggaat ttagattcgt atacatgcat ag 42
50 37 DNA Artificial Sequence Description of Artificial Sequence
SYNTHETIC DNA 50 gtctgaaggt tactctgact tcgatcgtgg tgcttac 37 51 37
DNA Artificial Sequence Description of Artificial Sequence
SYNTHETIC DNA 51 gtgataacgt aagcaccacg atcgaagtca gagtaac 37 52 38
DNA Artificial Sequence Description of Artificial Sequence
SYNTHETIC DNA 52 gttatcacct tcattccgaa atcttggaac actgctcc 38 53 38
DNA Artificial Sequence Description of Artificial Sequence
SYNTHETIC DNA 53 ctttgtccgg agcagtgttc caagatttcg gaatgaag 38 54 38
DNA Artificial Sequence Description of Artificial Sequence
SYNTHETIC DNA 54 ggacaaagtt aaacagggtt ggccgtaatg aaagctta 38 55 34
DNA Artificial Sequence Description of Artificial Sequence
SYNTHETIC DNA 55 agcttaagct ttcattacgg ccaaccctgt ttaa 34 56 20 DNA
Artificial Sequence Description of Artificial Sequence SYNTHETIC
DNA 56 ttttcccagt cacgacgttg 20 57 21 DNA Artificial Sequence
Description of Artificial Sequence SYNTHETIC DNA 57 caggaaacag
ctatgaccat g 21 58 36 DNA Artificial Sequence Description of
Artificial Sequence SYNTHETIC DNA 58 taaggaggtt taaaatgtct
gacgatcgtg ttactc 36 59 21 DNA Artificial Sequence Description of
Artificial Sequence SYNTHETIC DNA 59 tacgccaagg ttgttaaccc a 21 60
5 PRT Artificial Sequence Description of Artificial Sequence
N-TERMINAL FRAGMENT 60 Ser Asp Asp Arg Val 1 5 61 15 DNA Artificial
Sequence Description of Artificial Sequence CODON FOR N-TERMINAL
FRAGMENT 61 tctgacgatc gtgtt 15 62 5 PRT Artificial Sequence
Description of Artificial Sequence N-TERMINAL FRAGMENT 62 Met Ser
Asp Asp Arg 1 5
* * * * *