U.S. patent application number 14/539562 was filed with the patent office on 2015-03-05 for method for producing protein by precipitation.
This patent application is currently assigned to AJIMOMOTO CO., INC.. The applicant listed for this patent is AJINOMOTO CO., INC.. Invention is credited to TERUHISA MANNEN, TAKAHIRO NONAKA, NORIKO TSURUI.
Application Number | 20150064745 14/539562 |
Document ID | / |
Family ID | 51354258 |
Filed Date | 2015-03-05 |
United States Patent
Application |
20150064745 |
Kind Code |
A1 |
NONAKA; TAKAHIRO ; et
al. |
March 5, 2015 |
METHOD FOR PRODUCING PROTEIN BY PRECIPITATION
Abstract
The present invention provides a method for producing a target
protein in the form of a fusion protein at a high recovery ratio.
The present invention relates to a method for producing a fusion
protein made of a protein having a self-assembly capability and a
target protein comprising the following steps (1) to (4): (1)
preparing a solution containing the fusion protein; (2) adjusting a
pH of the solution obtained in step (1) to such a pH that a
recovery ratio calculated according to the following equation is
10% or more, where the recovery ratio (%)=[an amount of the fusion
protein in a solution obtained in step (4)/{the amount of the
fusion protein in the solution obtained in step (4)+an amount of
the fusion protein in a solution after solid separation in step
(3)}].times.100; (3) separating a solid from the solution obtained
in step (2); and (4) dissolving the solid separated in step (3)
into a solution having a pH of 12 or below but higher than the pH
of the solution obtained in step (2) by 0.1 or more.
Inventors: |
NONAKA; TAKAHIRO;
(KAWASAKI-SHI, JP) ; MANNEN; TERUHISA;
(KAWASAKI-SHI, JP) ; TSURUI; NORIKO;
(KAWASAKI-SHI, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AJINOMOTO CO., INC. |
CHUO-KU |
|
JP |
|
|
Assignee: |
AJIMOMOTO CO., INC.
CHUO-KU
JP
|
Family ID: |
51354258 |
Appl. No.: |
14/539562 |
Filed: |
November 12, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
PCT/JP2014/053810 |
Feb 18, 2014 |
|
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14539562 |
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Current U.S.
Class: |
435/68.1 ;
530/418 |
Current CPC
Class: |
C07K 2319/00 20130101;
C07K 14/62 20130101; C07K 2319/50 20130101; C07K 14/32 20130101;
C07K 2319/70 20130101; C07K 1/34 20130101; C07K 1/36 20130101; C07K
14/34 20130101; C07K 14/635 20130101; C07K 1/30 20130101; C07K
14/815 20130101; C07K 14/00 20130101 |
Class at
Publication: |
435/68.1 ;
530/418 |
International
Class: |
C07K 1/30 20060101
C07K001/30; C07K 14/00 20060101 C07K014/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 18, 2013 |
JP |
2013-029397 |
Claims
1. A method for producing a fusion protein made of a protein having
a self-assembly capability and a target protein, comprising the
following steps (1) to (4): (1) preparing a solution containing the
fusion protein; (2) adjusting a pH of the solution obtained in step
(1) to such a pH that a recovery ratio calculated according to the
following equation is 10% or more, wherein the recovery ratio
(%)=[an amount of the fusion protein in a solution obtained in step
(4)/{the amount of the fusion protein in the solution obtained in
step (4)+an amount of the fusion protein in a solution after solid
separation in step (3)}].times.100; (3) separating a solid from the
solution obtained in step (2); and (4) dissolving the solid
separated in step (3) into a solution having a pH of 12 or below
but higher than the pH of the solution obtained in step (2) by 0.1
or more.
2. The method according to claim 1, wherein the protein having a
self-assembly capability is a cell surface protein.
3. The method according to claim 2, wherein the cell surface
protein is a CspB mature protein or a portion thereof.
4. The method according to claim 3, wherein the CspB mature protein
or the portion thereof is any one of the following (a) and (b): (a)
a protein consisting of an amino acid sequence of SEQ ID NO: 3; and
(b) a protein having a homology of 95% or more with the amino acid
sequence of SEQ ID NO: 3.
5. The method according to claim 3, wherein the portion of the CspB
mature protein is a sequence consisting of 6 to 250 amino acid
residues from the N-terminus of the CspB mature protein.
6. The method according to claim 5, wherein the portion of the CspB
mature protein is a sequence consisting of 6, 17, 50, or 250 amino
acid residues from the N-terminus of the CspB mature protein.
7. The method according to claim 1, wherein the number of amino
acid residues in the target protein is 10 to 1000.
8. The method according to claim 1, wherein an amino acid sequence
used for an enzymatic cleavage or a chemical cleavage is further
incorporated between the protein having a self-assembly capability
and the target protein.
9. The method according to claim 8, wherein the amino acid sequence
used for the enzymatic cleavage between the protein having a
self-assembly capability and the target protein is a ProTEV
protease recognition sequence, a trypsin recognition sequence, or a
Factor Xa protease recognition sequence.
10. The method according to claim 1, wherein the pH in step (2) is
9 or below.
11. The method according to claim 1, wherein the pH is adjusted in
step (2) using an acid selected from the group consisting of
sulfuric acid, hydrochloric acid, acetic acid, phosphoric acid, and
trifluoroacetic acid.
12. The method according to claim 1, wherein the separation in step
(3) is performed by centrifugation and/or membrane filtration.
13. The method according to claim 1, wherein the solution obtained
in step (1) is a supernatant of a culture solution of a coryneform
bacterium having a gene construct capable of expressing the fusion
protein.
14. The method according to claim 1, wherein the recovery ratio
specified in step (2) is 30% or more.
15. A method for producing a target protein, comprising the
following steps (1) to (5): (1) preparing a solution containing a
fusion protein made of a protein having a self-assembly capability
and the target protein, the fusion protein containing an amino acid
sequence used for an enzymatic cleavage or a chemical cleavage
between the protein having a self-assembly capability and the
target protein; (2) adjusting a pH of the solution obtained in step
(1) to such a pH that a recovery ratio calculated according to the
following equation is 10% or more, wherein the recovery ratio
(%)=[an amount of the fusion protein in a solution obtained in step
(4)/{the amount of the fusion protein in the solution obtained in
step (4)+an amount of the fusion protein in a solution after solid
separation in step (3)}].times.100; (3) separating a solid from the
solution obtained in step (2); (4) dissolving the solid separated
in step (3) into a solution having a pH of 12 or below but higher
than the pH of the solution obtained in step (2) by 0.1 or more;
and (5) enzymatically or chemically cleaving the fusion protein at
a site of the amino acid sequence between the protein having a
self-assembly capability and the target protein simultaneously with
step (4), during step (4), or after step (4).
16. The method according to claim 15, wherein the step of cleaving
the fusion protein is an enzymatically cleaving step.
17. The method according to claim 15, wherein the protein having a
self-assembly capability is a cell surface protein.
18. The method according to claim 17, wherein the cell surface
protein is a CspB mature protein or a portion thereof.
19. The method according to claim 18, wherein the CspB mature
protein or the portion thereof is any one of the following (a) and
(b): (a) a protein consisting of an amino acid sequence of SEQ ID
NO: 3; and (b) a protein having a homology of 95%, or more with the
amino acid sequence of SEQ ID NO: 3.
20. The method according to claim 18, wherein the portion of the
CspB mature protein is a sequence consisting of 6 to 250 amino acid
residues from the N-terminus of the CspB mature protein.
21. The method according to claim 20, wherein the portion of the
CspB mature protein is a sequence consisting of 6, 17, 50, or 250
amino acid residues from the N-terminus of the CspB mature
protein.
22. The method according to claim 15, wherein the number of amino
acid residues in the target protein is 10 to 1000.
23. The method according to claim 15, wherein the target protein is
teriparatide.
24. The method according to claim 15, wherein the target protein is
a bivalirudin intermediate represented by SEQ ID NO: 93.
25. The method according to claim 15, wherein the amino acid
sequence used for the enzymatic cleavage between the protein having
a self-assembly capability and the target protein is a ProTEV
protease recognition sequence, a trypsin recognition sequence, or a
Factor Xa protease recognition sequence.
26. The method according to claim 15, wherein the pH in step (2) is
9 or below.
27. The method according to claim 15, wherein the pH is adjusted in
step (2) using an acid selected from the group consisting of
sulfuric acid, hydrochloric acid, acetic acid, phosphoric acid, and
trifluoroacetic acid.
28. The method according to claim 15, wherein the separation in
step (3) is performed by centrifugation and/or membrane
filtration.
29. The method according to claim 15, wherein the solution obtained
in step (1) is a supernatant of a culture solution of a coryneform
bacterium having a gene construct capable of expressing the fusion
protein.
30. The method according to claim 15, comprising a step of (6)
purifying the fusion protein or the target protein after step (4)
and/or step (5).
31. The method according to claim 30, wherein step (6) is performed
by column chromatography.
32. The method according to claim 15, wherein the recovery ratio
specified in step (2) is 300 or more.
33. A method for forming and separating a solid of a fusion protein
made of a protein having a self-assembly capability and a target
protein, the method comprising: forming the solid by adjusting a pH
of a solution containing the fusion protein to 9 or below; and then
separating the solid.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of and claims the
benefits of priority to International Application No.
PCT/JP2014/053810, filed Feb. 18, 2014, which claims the benefits
of priority to Japanese Application No. 2013-029397, filed Feb. 18,
2013. The entire contents of these applications are incorporated
herein by reference.
TECHNICAL FIELD
[0002] The present invention relates to a method for producing a
protein by precipitation. More specifically, the present invention
relates to methods for producing a fusion protein and a target
protein by adjusting a pH of a solution containing the fusion
protein made of the target protein and a protein having a
self-assembly capability.
BACKGROUND ART
[0003] Proteins having a variety of functions are widely utilized
for commercial uses such as pharmaceutical products and industrial
enzymes, and also for research uses in which proteins are used to
elucidate various biological phenomena. Thus, proteins are
substances essential in improvements in quality of life and
scientific advancements. As means for producing these various
proteins in large quantities at a low cost with a high
reproducibility, techniques for producing proteins using
recombinant organisms and techniques for purifying the proteins
have been developed.
[0004] The techniques for producing proteins using recombinant
organisms utilize animal cells (Non-Patent Literature 1) and
microorganisms (Non-Patent Literature 1). The animal cells used
include CHO cells (Non-Patent Literature 2), while the
microorganisms used include Escherichia coli, yeasts (Non-Patent
Literature 3), and the like. For example, there is a protein
secretory production system using Corynebacterium glutamicum
(hereinafter may be abbreviated as C. glutamicum) as the
microorganism (Patent Literature 1).
[0005] Main methods for purifying proteins produced by recombinant
organisms include a method utilizing properties of a protein
itself, and a method by adding a sequence used for purification to
a protein and utilizing properties of the added sequence.
[0006] The method utilizing properties of a protein itself includes
chromatography and liquid-solid separation.
[0007] The chromatography uses chromatographic matrixes having
various properties. The chromatography utilizes an interaction
between a protein and a chromatographic matrix, or the molecular
sieving effect of the chromatographic matrix (Non-Patent Literature
4). The interaction between a protein and a chromatographic matrix
includes electrostatic interaction, hydrogen bond, hydrophobic
interaction, specific interaction, and the like (Non-Patent
Literatures 4 and 5).
[0008] The liquid-solid separation is a separation method
including: insolubilizing (i.e., making solid) a protein in a
solubilized state by changing the solution conditions, obtaining a
solid component by a simple process such as centrifugation, and
bringing the separated solid component into a solubilized state
again. Specific examples of the means for insolubilizing the
protein include isoelectric point precipitation (Non-Patent
Literature 6), salting out (Non-Patent Literature 7), precipitation
using an organic solvent (Non-Patent Literature 8), precipitation
using a water-soluble polymer (Non-Patent Literature 9), and the
like.
[0009] The isoelectric point precipitation utilizes a property in
which the solubility of a protein becomes lowest at an isoelectric
point thereof. The salting out, the precipitation using an organic
solvent, and the precipitation using a water-soluble polymer
utilize a property in which the solubility of a protein is
decreased in the presence of the salt, the organic solvent, or the
water-soluble polymer, each of which is at a high concentration.
Another insolubilizing means is protein aggregation (Non-Patent
Literatures 10 and 11). The protein aggregation may be particularly
effective means when it is possible to select conditions for
aggregating a protein other than a protein to be purified while
leaving the protein to be purified in a solubilized state.
[0010] The method by adding a sequence used for purification to a
protein and utilizing properties of the added sequence includes a
method utilizing properties of the added sequence itself, and a
method utilizing an interaction between the added sequence and a
substance other than the added sequence.
[0011] The method utilizing properties of the added sequence itself
includes a method utilizing elastin, which undergoes a phase
transition to become insoluble with the temperature change
(Non-Patent Literature 12), a method utilizing MISTIC, which forms
a soluble assembly with the pH change (Patent Literature 2), and
the like.
[0012] In the method utilizing an interaction between the added
sequence and a substance other than the added sequence, the
substance other than the added sequence is often disposed on a
chromatographic matrix (Non-Patent Literature 13). The interaction
between the added sequence and the substance other than the added
sequence includes electrostatic interaction, hydrogen bond,
hydrophobic interaction, specific interaction, and the like.
CITATION LIST
Patent Literatures
[0013] Patent Literature 1: International Patent Application
Publication No. WO2005/103278 [0014] Patent Literature 2: European
Patent Application Publication No. 2423217
Non-Patent Literature
[0014] [0015] Non-Patent Literature 1: Demain A L, and Vaishnav P.,
Production of recombinant proteins by microbes and higher
organisms. Biotechnol Adv. 2009 May-June; 27 (3): 297-306. Epub
2009 Jan. 31. Review. [0016] Non-Patent Literature 2: Omasa T,
Onitsuka M, and Kim W D., Cell engineering and cultivation of
chinese hamster ovary (CHO) cells. Curr Pharm Biotechnol. 2010
April; 11 (3): 233-40. Review. [0017] Non-Patent Literature 3:
Mattanovich D, Branduardi P, Dato L, Gasser B, Sauer M, and Porro
D., Recombinant protein production in yeasts. Methods Mol. Biol.
2012; 824: 329-58. Review. [0018] Non-Patent Literature 4: Watanabe
E, Tsoka S, and Asenjo JA., Selection of chromatographic protein
purification operations based on physicochemical properties. Ann N
Y Acad Sci. 1994 May 2; 721: 348-64. [0019] Non-Patent Literature
5: Hober S, Nord K, and Linhult M., Protein A chromatography for
antibody purification. J Chromatogr B Analyt Technol Biomed Life
Sci. 2007 Mar. 15; 848 (1): 40-7. Epub 2006 Oct. 9. Review. [0020]
Non-Patent Literature 6: Garcia, F. A. P., Protein precipitation.
Recovery Processes Biol. Mater. (1993), 355-67. [0021] Non-Patent
Literature 7: A. A. Green: J. Biol. Chem., 95, 47 (1932) [0022]
Non-Patent Literature 8: McCord J M, and Fridovich I., Superoxide
dismutase. An enzymic function for erythrocuprein (hemocuprein). J
Biol. Chem. 1969 Nov. 25; 244 (22): 6049-55. [0023] Non-Patent
Literature 9: Ingham K C., Protein precipitation with polyethylene
glycol. Methods Enzymol. 1984; 104: 351-6. [0024] Non-Patent
Literature 10: Cromwell M E, Hilario E, and Jacobson F., Protein
aggregation and bioprocessing. AAPS J. 2006 Sep. 15; 8 (3): E572-9
[0025] Non-Patent Literature 11: Mahler H C, Friess W, Grauschopf
U, and Kiese S., J Pharm Sci. 2009 September; 98 (9): 2909-34.
Protein aggregation: pathways, induction factors and analysis.
[0026] Non-Patent Literature 12: Hassouneh W, Christensen T, and
Chilkoti A., Elastin-like polypeptides as a purification tag for
recombinant proteins. Curr Protoc Protein Sci. 2010 August; Chapter
6: Unit 6.11.
[0027] Non-Patent Literature 13: Smyth D R, Mrozkiewicz M K,
McGrath W J, Listwan P, and Kobe B., Crystal structures of fusion
proteins with large-affinity tags. Protein Sci. 2003 July; 12 (7):
1313-22.
SUMMARY OF INVENTION
Technical Problems
[0028] In the method utilizing properties of a protein itself, a
purification process is constructed for each target protein in
accordance with properties of the target protein. Hence, it is
generally impossible to employ a purification process for a certain
protein directly as a purification process for another protein.
[0029] On the other hand, the method by adding a sequence used for
purification to a protein and utilizing properties of the added
sequence can be said to be a widely applicable method, because the
same or similar purification process using an added sequence of the
same kind can be employed to purify multiple proteins in many
cases.
[0030] Nevertheless, such a method utilizing properties of an added
sequence also has problems: for example, the method utilizing
elastin cannot be employed for a target protein that is likely to
be inactivated by heat; and the method utilizing MISTIC has
difficulty separating an assembly formed.
Solution to Problems
[0031] The present inventors have earnestly studied to solve the
above-described problems. As a result, the inventors have
unexpectedly found out that when a protein having a self-assembly
capability is used as a sequence to be added, a fusion protein made
of the added sequence and the target protein has such a property
that the fusion protein is reversibly changeable between a
solubilized state and an insolubilized state in a solution in a pH
dependent manner. The present invention has been made based on this
finding.
[0032] Specifically, the present invention relates to the following
[1] to [33].
[1] A method for producing a fusion protein made of a protein
having a self-assembly capability and a target protein, comprising
the following steps (1) to (4):
[0033] (1) preparing a solution containing the fusion protein;
[0034] (2) adjusting a pH of the solution obtained in step (1) to
such a pH that a recovery ratio calculated according to the
following equation is 10% or more, where
the recovery ratio (%)=[an amount of the fusion protein in a
solution obtained in step (4)/{the amount of the fusion protein in
the solution obtained in step (4)+an amount of the fusion protein
in a solution after solid separation in step (3)}].times.100;
[0035] (3) separating a solid from the solution obtained in step
(2); and
[0036] (4) dissolving the solid separated in step (3) into a
solution having a pH of 12 or below but higher than the pH of the
solution obtained in step (2) by 0.1 or more.
[2] The method according to [1], in which the protein having a
self-assembly capability is a cell surface protein. [3] The method
according to [2], in which the cell surface protein is a CspB
mature protein or a portion thereof. [4] The method according to
[3], in which the CspB mature protein or the portion thereof is any
one of the following (a) and (b):
[0037] (a) a protein consisting of an amino acid sequence of SEQ ID
NO: 3; and
[0038] (b) a protein having a homology of 95% or more with the
amino acid sequence of SEQ ID NO: 3.
[5] The method according to [3], in which the portion of the CspB
mature protein is a sequence consisting of 6 to 250 amino acid
residues from the N-terminus of the CspB mature protein. [6] The
method according to [5], in which the portion of the CspB mature
protein is a sequence consisting of 6, 17, 50, or 250 amino acid
residues from the N-terminus of the CspB mature protein. [7] The
method according to any one of [1] to [6], in which the number of
amino acid residues in the target protein is 10 to 1000. [8] The
method according to any one of [1] to [7], in which an amino acid
sequence used for an enzymatic cleavage or a chemical cleavage is
further incorporated between the protein having a self-assembly
capability and the target protein. [9] The method according to [8],
in which the amino acid sequence used for the enzymatic cleavage
between the protein having a self-assembly capability and the
target protein is a ProTEV protease recognition sequence, a trypsin
recognition sequence, or a Factor Xa protease recognition sequence.
[10] The method according to any one of [1] to [9], in which the pH
in step (2) is 9 or below. [11] The method according to any one of
[1] to [10], in which the pH is adjusted in step (2) using an acid
selected from the group consisting of sulfuric acid, hydrochloric
acid, acetic acid, phosphoric acid, and trifluoroacetic acid. [12]
The method according to any one of [1] to [11], in which the
separation in step (3) is performed by centrifugation and/or
membrane filtration. [13] The method according to any one of [1] to
[12], in which the solution obtained in step (1) is a supernatant
of a culture solution of a coryneform bacterium having a gene
construct capable of expressing the fusion protein. [14] The method
according to any one of [1] to [13], in which the recovery ratio
specified in step (2) is 30% or more. [15] A method for producing a
target protein, comprising the following steps (1) to (5):
[0039] (1) preparing a solution containing a fusion protein made of
a protein having a self-assembly capability and the target protein,
the fusion protein containing an amino acid sequence used for an
enzymatic cleavage or a chemical cleavage between the protein
having a self-assembly capability and the target protein;
[0040] (2) adjusting a pH of the solution obtained in step (1) to
such a pH that a recovery ratio calculated according to the
following equation is 10% or more, where
the recovery ratio (%)=[an amount of the fusion protein in a
solution obtained in step (4)/{the amount of the fusion protein in
the solution obtained in step (4)+an amount of the fusion protein
in a solution after solid separation in step (3)}].times.100;
[0041] (3) separating a solid from the solution obtained in step
(2);
[0042] (4) dissolving the solid separated in step (3) into a
solution having a pH of 12 or below but higher than the pH of the
solution obtained in step (2) by 0.1 or more; and
[0043] (5) enzymatically or chemically cleaving the fusion protein
at a site of the amino acid sequence between the protein having a
self-assembly capability and the target protein simultaneously with
step (4), during step (4), or after step (4).
[16] The method according to [15], in which the step of cleaving
the fusion protein is an enzymatically cleaving step. [17] The
method according to [15], in which the protein having a
self-assembly capability is a cell surface protein. [18] The method
according to [17], in which the cell surface protein is a CspB
mature protein or a portion thereof. [19] The method according to
[18], in which the CspB mature protein or the portion thereof is
any one of the following (a) and (b):
[0044] (a) a protein consisting of an amino acid sequence of SEQ ID
NO: 3; and
[0045] (b) a protein having a homology of 95% or more with the
amino acid sequence of SEQ ID NO: 3.
[20] The method according to [18], in which the portion of the CspB
mature protein is a sequence consisting of 6 to 250 amino acid
residues from the N-terminus of the CspB mature protein. [21] The
method according to [20], in which the portion of the CspB mature
protein is a sequence consisting of 6, 17, 50, or 250 amino acid
residues from the N-terminus of the CspB mature protein. [22] The
method according to any one of [15] to [21], in which the number of
amino acid residues in the target protein is 10 to 1000. [23] The
method according to any one of [15] to [22], in which the target
protein is teriparatide. [24] The method according to any one of
[15] to [22], in which the target protein is a bivalirudin
intermediate represented by SEQ ID NO: 93. [25] The method
according to any one of [15] to [24], in which the amino acid
sequence used for the enzymatic cleavage between the protein having
a self-assembly capability and the target protein is a ProTEV
protease recognition sequence, a trypsin recognition sequence, or a
Factor Xa protease recognition sequence. [26] The method according
to any one of [15] to [25], in which the pH in step (2) is 9 or
below. [27] The method according to any one of [15] to [26], in
which the pH is adjusted in step (2) using an acid selected from
the group consisting of sulfuric acid, hydrochloric acid, acetic
acid, phosphoric acid, and trifluoroacetic acid. [28] The method
according to any one of [15] to [27], in which the separation in
step (3) is performed by centrifugation and/or membrane filtration.
[29] The method according to any one of [15] to [28], in which the
solution obtained in step (1) is a supernatant of a culture
solution of a coryneform bacterium having a gene construct capable
of expressing the fusion protein. [30] The method according to any
one of [15] to [29], comprising a step of (6) purifying the fusion
protein or the target protein after step (4) and/or step (5). [31]
The method according to [30], in which step (6) is performed by
column chromatography. [32] The method according to anyone of [15]
to [31], in which the recovery ratio specified in step (2) is 30%
or more. [33] A method for forming and separating a solid of a
fusion protein made of a protein having a self-assembly capability
and a target protein, the method comprising:
[0046] forming the solid by adjusting a pH of a solution containing
the fusion protein to 9 or below; and
[0047] then separating the solid.
Advantageous Effects of Invention
[0048] As described in Examples later, the present invention makes
it possible to obtain a fusion protein containing a target protein
or the target protein from a solution containing the fusion protein
easily at a high recovery ratio.
BRIEF DESCRIPTION OF DRAWINGS
[0049] FIG. 1-A shows the relation between the pH of "pH-adjusted
culture solutions" and the recovery ratio of a fusion protein
CspB50TEV-Teriparatide (abbreviated as 50-Teri) in Example 1.
[0050] FIG. 1-B shows the pH of the "pH-adjusted culture
solutions," photographs of band portions of the fusion protein
50-Teri in images of electrophoresis of "supernatants of the
pH-adjusted culture solutions" and the corresponding
"precipitate-dissolved solutions," and the recovery ratio in
Example 1.
[0051] FIG. 1-2A shows the relation between the pH of "pH-adjusted
culture solutions" and the recovery ratio of a fusion protein
50-Teri in Example 1-2.
[0052] FIG. 1-2B shows images of electrophoresis of the
"pH-adjusted culture solutions" and the corresponding
"precipitate-dissolved solutions," and photographs of extracted
band portions of the fusion protein 50-Teri in Example 1-2.
[0053] FIG. 1-2C shows chromatograms obtained by subjecting the
"supernatant of the pH-adjusted culture solution" having a pH
adjusted to 4.9 and the corresponding "precipitate-dissolved
solution" to reversed-phase HPLC in Example 1-2.
[0054] FIG. 1-2D shows chromatograms obtained by subjecting the
"precipitate-dissolved solution," which was prepared from the
"pH-adjusted culture solution" having a pH adjusted to 4.9, an
"enzymatic cleavage solution," and "standard Teriparatide" to
reversed-phase HPLC in Example 1-2.
[0055] FIG. 1-2E shows a chart of amass spectrum obtained by
subjecting a "purified substance," which was prepared by purifying
the "pH-adjusted culture solution" having a pH adjusted to 4.9, and
"standard Teriparatide" to mass spectrometry in Example 1-2.
[0056] FIG. 2-A shows the relation between the pH of "pH-adjusted
culture solutions" and the recovery ratio of a fusion protein
CspB50Lys-Bivalirudin18 (abbreviated as 50-Biva18) in Example
2.
[0057] FIG. 2-B shows the pH of the "pH-adjusted culture
solutions," photographs of band portions of the fusion protein
50-Biva18 in images of electrophoresis of "supernatants of the
pH-adjusted culture solutions" and the corresponding
"precipitate-dissolved solutions," and the recovery ratio in
Example 2.
[0058] FIG. 2-C shows chromatograms obtained by subjecting the
"supernatant of the pH-adjusted culture solution" having a pH
adjusted to 2.9 and the corresponding "precipitate-dissolved
solution" to reversed-phase HPLC in Example 2.
[0059] FIG. 2-D shows chromatograms obtained by subjecting the
"precipitate-dissolved solution," which was prepared from the
"pH-adjusted culture solution" having a pH adjusted to 2.9, and an
"enzymatic cleavage solution" to reversed-phase HPLC in Example
2.
[0060] FIG. 2-E shows amass (upper left part) calculated from the
amino acid sequence of Biva18, a theoretical mass spectrum (upper
right part) of the calculated mass and a chart (lower part) of a
mass spectrum obtained by subjecting a "purified substance," which
was prepared by purifying the "pH-adjusted culture solution" having
a pH adjusted to 2.9, to mass spectrometry in Example 2.
[0061] FIG. 2-F shows a chromatogram obtained by subjecting an
"enzymatic cleavage solution" prepared from the "pH-adjusted
culture solution" having a pH adjusted to 2.9 to strong anion
exchange resin chromatography in Example 2.
[0062] FIG. 2-G shows a chromatogram obtained by subjecting an
eluate (around 95% B) to reversed-phase HPLC, the eluate prepared
by subjecting the "enzymatic cleavage solution" prepared from the
"pH-adjusted culture solution" having a pH adjusted to 2.9 to
strong anion exchange resin chromatography in Example 2.
[0063] FIG. 3-A shows the relation between the pH of "pH-adjusted
culture solutions" and the recovery ratio of a fusion protein
CspB50TEV-Proinsulin (abbreviated as 50-PIns) in Example 3.
[0064] FIG. 3-B shows the pH of "pH-adjusted culture solutions,"
photographs of band portions of the fusion protein 50-PIns in
images of electrophoresis of "supernatants of the pH-adjusted
culture solutions" and the corresponding "precipitate-dissolved
solutions," and the recovery ratio in Example 3.
[0065] FIG. 4-A shows the relation between the pH of "pH-adjusted
culture solutions" and the recovery ratio of proinsulin
(abbreviated as PIns) in Comparative Example 1.
[0066] FIG. 4-B shows the pH of the "pH-adjusted culture
solutions," photographs of band portions of PIns in images of
electrophoresis of "supernatants of the pH-adjusted culture
solutions" and the corresponding "precipitate-dissolved solutions,"
and the recovery ratio in Comparative Example 1.
[0067] FIG. 5-A shows the relation between the pH of "pH-adjusted
culture solutions" and the recovery ratio of a fusion protein
CspB250TEV-Proinsulin (abbreviated as 250-PIns) in Example 4.
[0068] FIG. 5-B shows the pH of the "pH-adjusted culture
solutions," photographs of band portions of the fusion protein
250-PIns in images of electrophoresis of "supernatants of the
pH-adjusted culture solutions" and the corresponding
"precipitate-dissolved solutions," and the recovery ratio in
Example 4.
[0069] FIG. 6-A shows the relation between the pH of "pH-adjusted
culture solutions" and the recovery ratio of a fusion protein
CspB17TEV-Proinsulin (abbreviated as 17-PIns) in Example 5.
[0070] FIG. 6-B shows the pH of the "pH-adjusted culture
solutions," photographs of band portions of the fusion protein
17-PIns in images of electrophoresis of "supernatants of the
pH-adjusted culture solutions" and the corresponding
"precipitate-dissolved solutions," and the recovery ratio in
Example 5.
[0071] FIG. 7-A shows the relation between the pH of "pH-adjusted
culture solutions" and the recovery ratio of a fusion protein
CspB6TEV-Proinsulin (abbreviated as 6-PIns) in Example 6.
[0072] FIG. 7-B shows the pH of the "pH-adjusted culture solution,"
photographs of band portions of the fusion protein 6-PIns in images
of electrophoresis of "supernatants of the pH-adjusted culture
solutions" and the corresponding "precipitate-dissolved solutions,"
and the recovery ratio in Example 6.
[0073] FIG. 8-A shows the relation between the pH of "pH-adjusted
culture solutions" and the recovery ratio of a fusion protein
CspB50TEV-Teriparatide (abbreviated as 50-Teri) in Example 7.
[0074] FIG. 8-B shows the pH of the "pH-adjusted culture
solutions," photographs of band portions of the fusion protein
50-Teri in images of electrophoresis of "supernatants of the
pH-adjusted culture solutions" and the corresponding
"precipitate-dissolved solutions," and the recovery ratio in
Example 7.
[0075] FIG. 9-A shows the relation between the pH of "pH-adjusted
culture solutions" and the recovery ratio of a fusion protein
CspB50TEV-Teriparatide (abbreviated as 50-Teri) in Example 8.
[0076] FIG. 9-B shows the pH of the "pH-adjusted culture
solutions," photographs of band portions of the fusion protein
50-Teri in images of electrophoresis of "supernatants of the
pH-adjusted culture solutions" and the corresponding
"precipitate-dissolved solutions," and the recovery ratio in
Example 8.
[0077] FIG. 10-A shows chromatograms obtained by subjecting the
"microbial cell-removed culture solution" (corresponding to
"solution obtained in step (1)") and the "precipitate-dissolved
solution" (corresponding to "solution obtained in step (4)") in
Example 9 to reversed-phase HPLC (measuring wavelength: 280
nm).
[0078] FIG. 10-B shows chromatograms obtained by subjecting the
"microbial cell-removed culture solution" (corresponding to
"solution obtained in step (1)") and the "precipitate-dissolved
solution" (corresponding to "solution obtained in step (4)") in
Example 9 to reversed-phase HPLC (measuring wavelength: 220
nm).
DESCRIPTION OF EMBODIMENTS
[0079] A production method of the present invention is similar to
one type of a method by adding a sequence used for purification
(hereinafter may also be sometimes referred to as "added sequence")
to a protein and utilizing properties of the added sequence, that
is, a method utilizing the properties of the added sequence itself.
In the present invention, a sequence of a protein having a
self-assembly capability is used as the added sequence.
[0080] An embodiment of the present invention is a method for
producing a fusion protein made of a protein having a self-assembly
capability and a target protein, comprising the following steps (1)
to (4):
[0081] (1) preparing a solution containing the fusion protein;
[0082] (2) adjusting a pH of the solution obtained in step (1) to
such a pH that a recovery ratio calculated according to the
following equation is 10% or more, where
the recovery ratio (%)=[an amount of the fusion protein in a
solution obtained in step (4)/{the amount of the fusion protein in
the solution obtained in step (4)+an amount of the fusion protein
in a solution after solid separation in step (3)}].times.100;
[0083] (3) separating a solid from the solution obtained in step
(2); and
[0084] (4) dissolving the solid separated in step (3) into a
solution having a pH of 12 or below but higher than the pH of the
solution obtained in step (2) by 0.1 or more.
[0085] In step (1) of the production method of the present
invention, a solution containing a fusion protein made of a protein
having a self-assembly capability and a target protein is
prepared.
[0086] The "fusion protein" is composed of the "protein having a
self-assembly capability" and the "target protein". The "protein
having a self-assembly capability" may be located upstream (the
N-terminal side) or downstream (the C-terminal side) of the "target
protein." Note that, as will be described later, the fusion protein
may contain an "amino acid sequence used for a cleavage" between
the amino acid sequence of the "protein having a self-assembly
capability" and the amino acid sequence of the "target
protein."
[0087] The "self-assembly capability" refers to an ability of a
protein itself to assemble under appropriate environmental
conditions to thereby form a physiologically meaningful
higher-order structure.
[0088] The "protein having a self-assembly capability" may be a
full length sequence or a partial sequence, as long as the
self-assembly capability is retained.
[0089] The size (the number of amino acid residues) of the "protein
having a self-assembly capability" is not particularly limited, as
long as the self-assembly capability is retained. The number of the
amino acid residues is preferably 5 to 1000 amino acids, more
preferably 5 to 700 amino acids, and further preferably 5 to 500
amino acids.
[0090] The "protein having a self-assembly capability" may be a
variant of a naturally-occurring protein, as long as the
self-assembly capability is retained. For example, in the amino
acid sequence of a naturally-occurring protein, one or more amino
acids may be substituted, deleted, inserted, or added. The phrase
"one or more" means preferably 1 to 20, more preferably 1 to 10,
and further preferably 1 to 5, although the number varies depending
on the position of the amino acid residues in the conformation of
the protein and the type of the amino acid residues.
[0091] The substitution, deletion, insertion, or addition of the
amino acid may be a conservative mutation such that the
self-assembly capability of the protein is normally maintained. The
conservative mutation is typically a conservative substitution. The
conservative substitution is a mutation by which: Phe, Trp, and Tyr
are substituted for each other in a case where the substitution
site is an aromatic amino acid; Leu, Ile, and Val are substituted
for each other in a case where the substitution site is a
hydrophobic amino acid; Gln and Asn are substituted for each other
in a case of a polar amino acid; Lys, Arg, and His are substituted
for each other in a case of a basic amino acid; Asp and Glu are
substituted for each other in a case of an acidic amino acid; and
Ser and Thr are substituted for each other in a case of an amino
acid having a hydroxyl group. The substitution regarded as the
conservative substitution specifically includes a substitution of
Ala with Ser or Thr, a substitution of Arg with Gln, His, or Lys, a
substitution of Asn with Glu, Gln, Lys, His, or Asp, a substitution
of Asp with Asn, Glu, or Gln, a substitution of Cys with Ser or
Ala, a substitution of Gln with Asn, Glu, Lys, His, Asp, or Arg, a
substitution of Glu with Gly, Asn, Gln, Lys, or Asp, a substitution
of Gly with Pro, a substitution of His with Asn, Lys, Gln, Arg, or
Tyr, a substitution of Ile with Leu, Met, Val, or Phe, a
substitution of Leu with Ile, Met, Val, or Phe, a substitution of
Lys with Asn, Glu, Gln, His, or Arg, a substitution of Met with
Ile, Leu, Val, or Phe, a substitution of Phe with Trp, Tyr, Met,
Ile, or Leu, a substitution of Ser with Thr or Ala, a substitution
of Thr with Ser or Ala, a substitution of Trp with Phe or Tyr, a
substitution of Tyr with His, Phe, or Trp, and a substitution of
Val with Met, Ile, or Leu. Additionally, the substitution,
deletion, insertion, or addition of the amino acid includes those
caused in a naturally-occurring variant based on individual
differences in a bacterium from which a gene encoding the protein
is derived, differences in species, or the like.
[0092] The variant of a naturally-occurring protein may have a
homology of 95% or more, further preferably 97% or more, and
particularly preferably 99% or more, with the entire amino acid
sequence of the naturally-occurring protein, as long as the variant
has a self-assembly capability. Note that, herein, the "homology"
may also mean an "identity".
[0093] Moreover, the gene encoding the "protein having a
self-assembly capability" may be a DNA hybridizing under stringent
conditions with a probe that can be prepared from a known gene
sequence, for example, a sequence complementary to all or part of
the base sequence, as long as the encoded protein has a
self-assembly capability. The "stringent conditions" refer to
conditions under which a so-called specific hybrid is formed, but a
non-specific hybrid is not formed. Examples thereof include
conditions under which DNAs having a high homology with each other,
for example DNAs having a homology of 80% or more, preferably 90%
or more, more preferably 95% or more, further preferably 97% or
more, and particularly preferably 99% or more hybridize with each
other, but DNAs having lower homology than these do not hybridize
with each other; and conditions under which washing is performed
once, preferably two or three times, at a temperature and salt
concentrations of ordinal southern hybridization washing
conditions, that is, 60.degree. C., 1.times.SSC, 0.1% SDS,
preferably 60.degree. C., 0.1.times.SSC, 0.1% SDS, and more
preferably 68.degree. C., 0.1.times.SSC, 0.1% SDS.
[0094] The probe used in the hybridization may be a part of the
sequence complementary to the gene. Such a probe can be produced by
PCR using as primers oligonucleotides prepared based on a known
gene sequence, and using as a template a DNA fragment containing
the base sequences of these. For example, when a DNA fragment of
approximately 300 bp in length is used as the probe, the
hybridization washing conditions include 50.degree. C.,
2.times.SSC, 0.1% SDS.
[0095] The gene encoding the "protein having a self-assembly
capability" can be used in a naturally-occurring form, or may be
modified so as to have optimum codons in accordance with the codon
usage in a host to be used.
[0096] Specific examples of the "protein having a self-assembly
capability" include cell surface proteins, viral envelope proteins,
various motor proteins, and the like.
[0097] A cell surface protein is a component protein of a cell
surface structure, called an S-layer, widely found in bacteria, and
is known to self-assemble and form a layered structure under
physiological conditions (Ilk N, Egelseer E M, and Sleytr U B.
S-layer fusion proteins--construction principles and applications.
Curr Opin Biotechnol. 2011 December; 22 (6): 824-31. Epub 2011 Jun.
21.).
[0098] Specific examples of the cell surface proteins include PS1
and CspB (PS2) derived from a coryneform bacterium C. glutamicum
(Published Japanese Translation of PCT Internal Application No.
(JP-A) Hei 6-502548), SlpA (CspA) derived from Corynebacterium
ammoniagenes (Japanese Patent Application Publication No. (JP-A)
Hei 10-108675), and the like.
[0099] Among these, CspB (PS2) (499 amino acid residues) is
preferable. Note that CspB is the same as PS2.
[0100] CspB is a cell surface protein found in C. glutamicum
(Peyret J L, Bayan N, Joliff G, Gulik-Krzywicki T, Mathieu L,
Schechter E, and Leblon G., Characterization of the cspB gene
encoding PS2, an ordered surface-layer protein in Corynebacterium
glutamicum. Mol. Microbiol. 1993 July; 9 (1): 97-109).
[0101] As to CspB, it has been found out in various types of C.
glutamicum that treating the microbial cells with a solution
containing a surfactant sodium dodecyl sulfate (SDS) disrupts the
self-assembled layered structure, and CspB is extracted into the
SDS solution (Hansmeier N, Bartels F W, Ros R, Anselmetti D, Tauch
A, Puhler A, and Kalinowski J. Classification of hyper-variable
Corynebacterium glutamicum surface-layer proteins by sequence
analyses and atomic force microscopy. J Biotechnol. 2004 Aug. 26;
112 (1-2): 177-93).
[0102] A specific example of CspB includes CspB of C. glutamicum
ATCC13869. The base sequence of a cspB gene of C. glutamicum
ATCC13869 is shown in SEQ ID NO: 1, and the amino acid sequence of
a CspB protein is shown in SEQ ID NO: 2. In the amino acid sequence
shown in SEQ ID NO: 2, amino acid residues at positions 1 to 30
correspond to a signal peptide, and amino acid residues at
positions 31 to 499 correspond to a CspB mature protein
(hereinafter may also be referred to as either "mature CspB" or
"CspB mature protein"). The amino acid sequence of the CspB mature
protein of C. glutamicum ATCC13869 wherein the 30 amino acid
residues of the signal peptide portion are excluded is shown in SEQ
ID NO: 3.
TABLE-US-00001 SEQ ID NO: 3 Gln Glu Thr Asn Pro Thr Phe Asn Ile Asn
Asn Gly Phe Asn Asp Ala Asp Gly Ser Thr Ile Gln Pro Val Glu Pro Val
Asn His Thr Glu Glu Thr Leu Arg Asp Leu Thr Asp Ser Thr Gly Ala Tyr
Leu Glu Glu Phe Gln Tyr Gly Asn Val Glu Glu Ile Val Glu Ala Tyr Leu
Gln Val Gln Ala Ser Ala Asp Gly Phe Asp Pro Ser Glu Gln Ala Ala Tyr
Glu Ala Phe Glu Ala Ala Arg Val Arg Ala Ser Gln Glu Leu Ala Ala Ser
Ala Glu Thr Ile Thr Lys Thr Arg Glu Ser Val Ala Tyr Ala Leu Lys Ala
Asp Arg Glu Ala Thr Ala Ala Phe Glu Ala Tyr Leu Ser Ala Leu Arg Gln
Val Ser Val Ile Asn Asp Leu Ile Ala Asp Ala Asn Ala Lys Asn Lys Thr
Asp Phe Ala Glu Ile Glu Leu Tyr Asp Val Leu Tyr Thr Asp Ala Asp Ile
Ser Gly Asp Ala Pro Leu Leu Ala Pro Ala Tyr Lys Glu Leu Lys Asp Leu
Gln Ala Glu Val Asp Ala Asp Phe Glu Trp Leu Gly Glu Phe Ala Ile Asp
Asn Asn Glu Asp Asn Tyr Val Ile Arg Thr His Ile Pro Ala Val Glu Ala
Leu Lys Ala Ala Ile Asp Ser Leu Val Asp Thr Val Glu Pro Leu Arg Ala
Asp Ala Ile Ala Lys Asn Ile Glu Ala Gln Lys Ser Asp Val Leu Val Pro
Gln Leu Phe Leu Glu Arg Ala Thr Ala Gln Arg Asp Thr Leu Arg Val Val
Glu Ala Ile Phe Ser Thr Ser Ala Arg Tyr Val Glu Leu Tyr Glu Asn Val
Glu Asn Val Asn Val Glu Asn Lys Thr Leu Arg Gln His Tyr Ser Ser Leu
Ile Pro Asn Leu Phe Ile Ala Ala Val Gly Asn Ile Asn Glu Leu Asn Asn
Ala Asp Gln Ala Ala Arg Glu Leu Phe Leu Asp Trp Asp Thr Asp Leu Thr
Thr Asn Asp Glu Asp Glu Ala Tyr Tyr Gln Ala Lys Leu Asp Phe Ala Ile
Glu Thr Tyr Ala Lys Ile Leu Ile Asn Gly Glu Val Trp Gln Glu Pro Leu
Ala Tyr Val Gln Asn Leu Asp Ala Gly Ala Arg Gln Glu Ala Ala Asp Arg
Glu Ala Glu Arg Ala Ala Asp Ala Ala Tyr Arg Ala Glu Gln Leu Arg Ile
Ala Gln Glu Ala Ala Asp Ala Gln Lys Ala Leu Ala Glu Ala Leu Ala Asn
Ala Gly Asn Asn Asp Asn Gly Gly Asp Asn Ser Ser Asp Asp Lys Gly Thr
Gly Ser Ser Asp Ile Gly Thr Trp Gly Pro Phe Ala Ala Ile Ala Ala Ile
Ile Ala Ala Ile Ala Ala Ile Phe Pro Phe Leu Ser Gly Ile Val Lys
Phe
[0103] CspB used in the present invention is preferably a CspB
mature protein, more preferably a portion of the CspB mature
protein. Particularly, CspB used in the present invention
preferably is a sequence consisting of 6 to 250 amino acid residues
from the N-terminus of the CspB mature protein from the standpoint
of the recovery ratio of the fusion protein. The "sequence
consisting of 6 to 250 amino acid residues from the N-terminus" is
an amino acid sequence from an amino acid residue at position 1 at
the N-terminus to an amino acid residue at any one of positions 6
to 250 (any one of 6th to 250th amino acid residues from the same
N-terminus).
[0104] For example, in a case where the CspB mature protein is the
protein consisting of an amino acid sequence shown in SEQ ID NO: 3,
the "sequence consisting of 6 to 250 amino acid residues from the
N-terminus" is an amino acid sequence from an amino acid residue at
position 1 (the N-terminus) of SEQ ID NO: 3 to an amino acid
residue at any one of positions 6 to 250 (any one of 6th to 250th
amino acid residues from the same N-terminus).
[0105] Further preferably, CspB used in the present invention is a
sequence consisting of 6, 17, 50, or 250 amino acid residues from
the N-terminus of the CspB mature protein.
[0106] It should be noted that the phrase "position X (X is for
example 6, 17, 50, or 250)" in an amino acid sequence means the Xth
position from the N-terminus in the amino acid sequence, and an
amino acid residue at the N-terminus is an amino acid residue at
position 1. Namely, the positions of the aforementioned amino acid
residues show relative positions, and therefore the positions may
change slightly by a deletion, insertion, addition, or the like of
an amino acid.
[0107] For example, in a case where the CspB mature protein is the
protein consisting of an amino acid sequence shown in SEQ ID NO: 3,
the "amino acid residue at position 50 from the N-terminus" means
an amino acid residue corresponding to one at position 50 in SEQ ID
NO: 3. If one amino acid residue is deleted between position 49 and
the N-terminus, the 49th amino acid residue from the N-terminus is
the "amino acid residue at position 50 from the N-terminus." In
addition, if one amino acid residue is inserted between position 50
and the N-terminus, the 51st amino acid residue from the N-terminus
is the "amino acid residue at position 50 from the N-terminus."
[0108] The nucleic acid sequence of the cspB gene varies, depending
on the species to which a coryneform bacterium belongs or the
strain thereof. Thus, the cspB gene may be a variant of the nucleic
acid sequence, as long as the encoded protein has a self-assembly
capability. The variant of the cspB gene includes homologues of the
gene. The homologues of the cspB gene can be easily obtained, for
example, from a publicly-available database by a BLAST search or a
FASTA search using the above-described cspB gene (SEQ ID NO: 1) of
C. glutamicum ATCC13869 as a query sequence. The homologues of the
cspB gene can also be obtained by PCR using a chromosome of the
coryneform bacterium as a template, and using as primers
oligonucleotides prepared based on these known gene sequences.
[0109] The "target protein" includes any naturally-occurring
proteins derived from microorganisms, plants, animals, or viruses,
and proteins whose amino acid sequences are artificially designed,
without particular limitations.
[0110] In addition, the "target protein" may be a homologous
protein or a heterologous protein in relation to a host producing
the protein.
[0111] The target protein may be a monomeric protein or a
multimeric protein (multimer).
[0112] The multimeric protein refers to a protein existing as a
multimer composed of two or more subunits. In a multimer, each
subunit may be linked to the other(s) by a covalent bond such as a
disulfide bond, or linked by a non-covalent bond such as a hydrogen
bond or hydrophobic interaction, or linked by a combination of
these. The multimer may be a homomultimer composed of the same type
of subunits, or a heteromultimer composed of two or more types of
subunits. Incidentally, in a case where the multimeric protein is a
heteromultimer, at least one subunit among subunits composing the
multimer may be a heterologous protein. In other words, all the
subunits may be derived from different species, or only some
subunits may be derived from different species.
[0113] The target protein may be a naturally-occurring secretory
protein, or a naturally-occurring non-secretory protein. A
naturally-occurring secretory protein is preferable.
[0114] The number of types of the target protein produced according
to the present invention may be only one or may be two or more.
Moreover, in a case where the target protein is a heteromultimer,
only one type of subunits may be produced, or two or more types of
subunits may be produced.
[0115] The size (the number of amino acid residues) of the target
protein is not particularly limited, as long as it can be expressed
in a host to be used. The number of the amino acid residues is
preferably 10 to 1000, more preferably 10 to 500, and further
preferably 10 to 300.
[0116] The target protein may be a variant of the
naturally-occurring protein. The above description of the variant
related to the "protein having a self-assembly capability" can
apply to the target protein and a gene encoding the target
protein.
[0117] Further, the gene encoding the target protein may be
modified if necessary so as to have optimum codons in accordance
with the codon usage in a host.
[0118] Examples of the target protein include bioactive proteins,
receptor proteins, antigen proteins used as vaccines, and
enzymes.
[0119] Examples of the bioactive proteins include growth factors
(proliferative factors), hormones, cytokines, and antibody-related
molecules.
[0120] Specific examples of the growth factors (proliferative
factors) include epidermal growth factor (EGF), insulin-like growth
factor-1 (IGF-1), transforming growth factor (TGF), nerve growth
factor (NGF), brain-derived neurotrophic factor (BDNF), vesicular
endothelial growth factor (VEGF), granulocyte-colony stimulating
factor (G-CSF), granulocyte-macrophage-colony stimulating factor
(GM-CSF), platelet-derived growth factor (PDGF), erythropoietin
(EPO), thrombopoietin (TPO), acidic fibroblast growth factor (aFGF
or FGF1), basic fibroblast growth factor (bFGF or FGF2),
keratinocyte growth factors (KGF-1 or FGF7, KGF-2 or FGF10), and
hepatocyte growth factor (HGF).
[0121] Specific examples of the hormones include insulins,
glucagon, somatostatin, human growth hormones (hGHs), parathyroid
hormone (PTH), and calcitonin.
[0122] Specific examples of the cytokines include interleukins,
interferons, and tumor necrosis factors (TNFs).
[0123] Note that the growth factors (proliferative factors), the
hormones, and the cytokines do not necessarily have to be strictly
distinguished from each other. For example, the bioactive protein
may belong to any one group selected from the growth factors
(proliferative factors), the hormones, and the cytokines, or may
belong to multiple groups selected therefrom.
[0124] Furthermore, the bioactive protein may be a full length
protein or a portion thereof. An example of the portion of the
protein includes a portion having a physiological activity. A
specific example of the portion having a physiological activity
includes teriparatide consisting of 34 amino acid residues at the
N-terminus of matured parathyroid hormone (PTH).
[0125] Incidentally, the target protein may be a portion of a
bioactive protein, the portion not having a physiological activity.
In this case, after the target protein is obtained, a necessary
modification (for example, addition of an amino acid sequence) is
performed, and thus a bioactive protein can be obtained. A specific
example includes a peptide (Biva18) (SEQ ID NO: 93: Arg Pro Gly Gly
Gly Gly Asn Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu), which is
a portion (18 amino acid residues) of a bioactive protein
bivalirudin (20 amino acids).
[0126] Specific examples of the antibody-related molecules include
complete antibodies, Fab, F (ab'), F (ab').sub.2, Fc, a dimer
composed of a heavy chain (H chain) and a light chain (L chain), Fc
fusion proteins, heavy chains (H chains), and light chains (L
chains).
[0127] The receptor proteins are not particularly limited, and may
be, for example, receptor proteins for bioactive proteins or other
bioactive substances. Examples of the other bioactive substances
include neurotransmitters such as dopamine. Additionally, the
receptor proteins may be orphan receptors whose corresponding
ligands are unknown.
[0128] The antigen proteins used as vaccines are not particularly
limited, as long as an immune response can be elicited, and can be
selected as appropriate in accordance with a target of an intended
immune response.
[0129] Examples of the enzymes include transglutaminases,
proteases, endopeptidases, exopeptidases, aminopeptidases,
carboxypeptidases, collagenases, chitinases, and the like.
[0130] The target protein may be a protein (proprotein), to which a
pro-structural portion is added. In a case where the target protein
is such a proprotein, a mature protein can be obtained by cleaving
the pro-structural portion.
[0131] The cleaving of the pro-structural portion can be performed
simultaneously with step (4), during step (4), or after step (4) in
the production method of the present invention.
[0132] The phrase "simultaneously with step (4)" means that step
(4) and the cleaving of the pro-structural portion are
simultaneously performed using a solution obtained by adding in
advance a reagent (for example, a protease to be described below)
used for cleaving the pro-structural portion to a "solution having
a pH of 12 or below but higher than the pH of a solution obtained
in step (2) by 0.1 or more" used in step (4).
[0133] The phrase "during step (4)" means that step (4) is
initiated without adding the reagent used for cleaving the
pro-structural portion to the "solution having a pH of 12 or below
but higher than the pH of the solution obtained in step (2) by 0.1
or more" used in step (4), and then the reagent is added during
step (4) to cleave the pro-structural portion.
[0134] The phrase "after step (4)" means that after step (4) is
completed, the pro-structural portion is cleaved using the reagent
used for cleaving the pro-structural portion.
[0135] The pro-structural portion can be cleaved with, for example,
a protease. When a protease is used, from the viewpoint of the
activity of the finally obtained protein, the proprotein is
generally preferably cleaved at approximately the same position as
those of naturally-occurring proteins, and more preferably cleaved
at completely the same position as those of naturally-occurring
proteins so that the same mature protein as naturally-occurring
ones can be obtained. Thus, generally, the most preferable is a
specific protease that cleaves the proprotein at this position so
that the same protein as naturally-occurring mature proteins can be
obtained.
[0136] The protease includes ones commercially available such as
proTEV protease (manufactured by Promega Corporation), and ones
obtained from culture solutions of microorganisms such as a culture
solution of an actinobacterium. These proteases can be used in an
unpurified state, or may be used after purified to an appropriate
purity as necessary.
[0137] The fusion protein made of the protein having a
self-assembly capability and the target protein can be obtained by
expressing a gene construct for expressing the fusion protein in a
host.
[0138] The "host" is not particularly limited, as long as it is
capable of expressing the fusion protein. It is possible to use all
types of bacteria, microorganisms other than bacteria, insect
cells, animal cells, and the like.
[0139] In a case of using a host capable of accumulating the fusion
protein in its microbial cells, the cells are disrupted to prepare
the solution in step (1). A host capable of secreting the fusion
protein outside the cells is preferable because such a disruption
treatment is not necessary.
[0140] As the bacteria, for example, coryneform bacteria,
Escherichia coli, and the like can be used.
[0141] As the microorganisms other than bacteria, for example,
yeasts and the like can be used.
[0142] As the insect cells, Bombyx mori and the like can be used.
As the animal cells, CHO cells and the like can be used.
[0143] Among these, coryneform bacteria are preferable.
[0144] In the present invention, the "coryneform bacteria" are
aerobic gram-positive bacilli, and include Corynebacterium
bacteria, Brevibacterium bacteria, Microbacterium bacteria, and so
forth. The coryneform bacteria include bacteria which have
previously been classified into the genus Brevibacterium but are
presently united into the genus Corynebacterium (Int. J. Syst.
Bacteriol., 41, 255 (1991)).
[0145] Advantages of using coryneform bacteria include the facts
that: the amount of impurity proteins inherently secreted outside
the cells is extremely small in comparison with filamentous fungi,
yeasts, bacteria of the genus Bacillus, and the like, which have
been utilized for secretory production of target proteins, so that
simplification and omission of the purification step for secretory
production of a fusion protein can be expected; coryneform bacteria
grow well in a simple medium containing a sugar, ammonia, an
inorganic salt, and the like, and are thus excellent in terms of
medium cost, culturing method, culture productivity; and
others.
[0146] The species of the coryneform bacteria specifically include
the following: [0147] Corynebacterium acetoacidophilum, [0148]
Corynebacterium acetoglutamicum, [0149] Corynebacterium
alkanolyticum, [0150] Corynebacterium callunae, [0151]
Corynebacterium glutamicum, [0152] Corynebacterium lilium, [0153]
Corynebacterium melassecola, [0154] Corynebacterium
thermoaminogenes (Corynebacterium efficiens), [0155]
Corynebacterium herculis, [0156] Brevibacterium divaricatum, [0157]
Brevibacterium flavum, [0158] Brevibacterium immariophilum, [0159]
Brevibacterium lactofermentum (Corynebacterium glutamicum), [0160]
Brevibacterium roseum, [0161] Brevibacterium saccharolyticum,
[0162] Brevibacterium thiogenitalis, [0163] Corynebacterium
ammoniagenes, [0164] Brevibacterium album, [0165] Brevibacterium
cerinum, and
[0166] Microbacterium ammoniaphilum.
[0167] The strains of the coryneform bacteria specifically include
the following: [0168] Corynebacterium acetoacidophilum ATCC 13870,
[0169] Corynebacterium acetoglutamicum ATCC 15806, [0170]
Corynebacterium alkanolyticum ATCC 21511, [0171] Corynebacterium
callunae ATCC 15991, [0172] Corynebacterium glutamicum ATCC 13020,
ATCC 13032, ATCC 13060, ATCC 13869, FERM BP-734, [0173]
Corynebacterium lilium ATCC 15990, [0174] Corynebacterium
melassecola ATCC 17965, [0175] Corynebacterium thermoaminogenes
FERM BP-1539, [0176] Corynebacterium herculis ATCC 13868, [0177]
Brevibacterium divaricatum ATCC 14020, [0178] Brevibacterium flavum
ATCC 13826, ATCC 14067, FERM BP-2205, [0179] Brevibacterium
immariophilum ATCC 14068, [0180] Brevibacterium lactofermentum ATCC
13869, [0181] Brevibacterium roseum ATCC 13825, [0182]
Brevibacterium saccharolyticum ATCC 14066, [0183] Brevibacterium
thiogenitalis ATCC 19240, [0184] Brevibacterium ammoniagenes ATCC
6871, ATCC 6872, [0185] Brevibacterium album ATCC 15111, [0186]
Brevibacterium cerinum ATCC 15112, and [0187] Microbacterium
ammoniaphilum ATCC 15354.
[0188] C. glutamicum FERM BP-734 isolated as a streptomycin
(Sm)-resistant mutant strain from a wildtype strain C. glutamicum
ATCC13869 is expected to have a mutation in a functional gene
involved in protein secretion, and has an extremely high secretory
productivity of a heterologous protein, the amount of which
accumulated under optimum culture conditions is approximately 2 to
3 times larger than that of the parent strain (wildtype strain).
Thus, C. glutamicum FERM BP-734 is suitable as the host
bacterium.
[0189] Using the above-described coryneform bacterium as a parent
strain, a strain having an increased protein secretory productivity
may be selected by utilizing a mutagenesis or gene recombination
method and used as the host. For example, after a treatment with an
ultraviolet irradiation or a chemical mutagen such as
N-methyl-N'-nitrosoguanidine, a strain having an increased protein
secretory productivity can be selected.
[0190] Further, a strain modified from the selected strain in such
a manner as not to produce the cell surface protein is particularly
preferably used as the host because it is easy to purify a
heterologous protein secreted into a medium or to a microbial cell
surface layer. Such a modification can be carried out by
introducing a mutation into a coding region of the cell surface
protein or an expression regulatory region thereof on a chromosome
by a mutagenesis or gene recombination method. The coryneform
bacterium modified not to produce the cell surface protein includes
a C. glutamicum YDK010 strain that is a cell surface protein CspB
(PS2)-disrupted strain of C. glutamicum AJ12036 (FERM BP-734)
(WO2004/029254).
[0191] In a case where the protein having a self-assembly
capability is located upstream of the target protein, the "gene
construct for expressing the fusion protein" contains: a promoter
sequence which functions in the host; a nucleic acid sequence
encoding a signal peptide which functions in the host, the nucleic
acid sequence connected downstream of the promoter sequence; a
nucleic acid sequence encoding the protein having a self-assembly
capability, the nucleic acid sequence connected downstream of the
nucleic acid sequence encoding the signal peptide; and a nucleic
acid sequence encoding the target protein, the nucleic acid
sequence connected downstream of the nucleic acid sequence encoding
the protein having a self-assembly capability. Meanwhile, in a case
where the protein having a self-assembly capability is located
downstream of the target protein, the "gene construct for
expressing the fusion protein" contains: the promoter sequence
which functions in the host; the nucleic acid sequence encoding the
signal peptide which functions in the host, the nucleic acid
sequence connected downstream of the promoter sequence; the nucleic
acid sequence encoding the target protein, the nucleic acid
sequence connected downstream of the nucleic acid sequence encoding
the signal peptide; and the nucleic acid sequence encoding the
protein having a self-assembly capability, the nucleic acid
sequence connected downstream of the nucleic acid sequence encoding
the target protein. Note that in a case where the fusion protein is
accumulated in the microbial cells, the nucleic acid sequence
encoding the signal peptide is not necessary.
[0192] The nucleic acid sequence encoding the signal peptide should
be ligated downstream of the promoter sequence so that the signal
peptide is expressed under the control of the promoter.
[0193] In the case where the protein having a self-assembly
capability is located upstream of the target protein, the nucleic
acid sequence encoding the protein having a self-assembly
capability should be ligated downstream of the nucleic acid
sequence encoding the signal peptide so that the protein having a
self-assembly capability connected to the signal peptide can be
expressed. Further, the nucleic acid sequence encoding the target
protein should be ligated downstream of the nucleic acid sequence
encoding the protein having a self-assembly capability so that the
target protein connected to the protein having a self-assembly
capability can be expressed.
[0194] In the case where the protein having a self-assembly
capability is located downstream of the target protein, the nucleic
acid sequence encoding the target protein should be ligated
downstream of the nucleic acid sequence encoding the signal peptide
so that the target protein connected to the signal peptide can be
expressed. Further, the nucleic acid sequence encoding the protein
having a self-assembly capability should be ligated downstream of
the nucleic acid sequence encoding the target protein so that the
protein having a self-assembly capability connected to the target
protein can be expressed.
[0195] The gene construct may contain control sequences (an
operator, a terminator, and the like) at suitable positions so that
they can function to effectively express the fusion protein gene in
the host.
[0196] The "promoter" is not particularly limited, as long as the
promoter functions in the host to be used. The promoter may be
derived from the host, or of a heterologous origin.
[0197] For example, when a coryneform bacterium is used as the
host, the promoter is a promoter which functions in the coryneform
bacterium.
[0198] The "promoter which functions in the coryneform bacterium"
refers to a promoter having a promoter activity in the coryneform
bacterium.
[0199] Examples of the promoter derived from the coryneform
bacterium include promoters of genes of the cell surface proteins
PS1, CspB (may also be referred to as PS2), and SlpA (may also be
referred to as CspA), and promoters of various amino acid
biosynthesis genes.
[0200] Specific examples of the promoters of various amino acid
biosynthesis genes include promoters of a glutamate dehydrogenase
gene in a glutamic acid biosynthesis system, a glutamine synthetase
gene in a glutamine synthesis system, an aspartokinase gene in a
lysine biosynthesis system, a homoserine dehydrogenase gene in a
threonine biosynthesis system, an acetohydroxy acid synthetase gene
in isoleucine and valine biosynthesis systems, a 2-isopropyl malic
acid synthetase gene in a leucine biosynthesis system, a glutamate
kinase gene in proline and arginine biosynthesis systems, a
phosphoribosyl-ATP pyrophosphorylase gene in a histidine
biosynthesis system, a deoxy-arabino-heptulosonate phosphate (DAHP)
synthetase gene in biosynthesis systems of aromatic amino acids
such as tryptophan, tyrosine, and phenylalanine, and a
phosphoribosyl pyrophosphate (PRPP) amidotransferase gene, an
inosinic acid dehydrogenase gene, and a guanylic acid synthetase
gene in biosynthesis systems of nucleic acids such as inosinic acid
and guanylic acid.
[0201] Specific examples of the heterologous promoter in relation
to coryneform bacteria include promoters derived from E. coli, such
as a tac promoter, a lac promoter, a trp promoter, and an araBAD
promoter. Among these, a strong promoter such as a tac promoter is
preferable, and an inducible promoter such as an araBAD promoter is
also preferable.
[0202] By using various reporter genes, a native promoter in a
highly active form may be obtained and utilized. For example, by
making -35 or -10 region in a promoter region close to a consensus
sequence, the activity of the promoter can be increased
(International Patent Application Publication No. WO00/18935).
Examples of methods for evaluating promoter strength and strong
promoters are described in the paper by Goldstein et al.
(Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1,
105-128 (1995)) and so on. Further, a substitution, an insertion,
or a deletion of several nucleotides in a spacer region between a
ribosome binding site (RBS) and a start codon, particularly in a
sequence (5'-UTR) immediately upstream of the start codon, is known
to greatly influence the mRNA stability and translation efficiency,
and these can also be modified.
[0203] In the present invention, the host preferably produces the
fusion protein as a secretory protein.
[0204] Generally, it is known that a secretory protein is
translated as a preprotein (may also be referred to as prepeptide)
or a preproprotein (may also be referred to as prepropeptide), and
then becomes a mature protein by the subsequent processing.
Specifically, generally, after a secretory protein is translated as
a preprotein or a preproprotein, a pre-portion thereof, that is, a
signal peptide, is cleaved by a protease (generally called a signal
peptidase) to thus produce and secrete a mature protein or a
proprotein, while a pro-portion of the proprotein is further
cleaved by a protease to thus produce a mature protein.
[0205] Accordingly, in the present invention, a signal peptide is
utilized for the secretory production of the fusion protein by the
host. Note that, herein, the preprotein and the preproprotein of
the secretory protein are collectively referred to as a "secretory
protein precursor" in some cases.
[0206] The term "signal peptide" (hereinafter may also be sometimes
referred to as "signal sequence") refers to an amino acid sequence
present at the N-terminus of the secretory protein precursor, but
normally absent in naturally-occurring mature proteins.
[0207] The signal peptide used in the present invention is not
particularly limited, as long as the signal peptide functions in
the host. The signal peptide may be derived from the host, or of a
heterologous origin.
[0208] For example, when a coryneform bacterium is used as the
host, the signal peptide is a signal peptide which functions in the
coryneform bacterium.
[0209] The "signal peptide which functions in the coryneform
bacterium" refers to a peptide allowing the coryneform bacterium to
secrete the fusion protein when the peptide is ligated to the
N-terminus of the fusion protein.
[0210] The signal peptide which functions in the coryneform
bacterium is preferably a signal peptide of a secretory protein of
the host coryneform bacterium, more preferably a signal peptide of
a cell surface protein of the coryneform bacterium. The cell
surface protein of the coryneform bacterium include PS1 and CspB
(PS2) derived from C. glutamicum (JP-A Hei 6-502548), and SlpA
(CspA) derived from C. ammoniagenes (JP-A Hei 10-108675). The amino
acid sequence of the signal peptide of PS1 is shown in SEQ ID NO:
4, the amino acid sequence of the signal peptide of CspB (PS2) is
shown in SEQ ID NO: 5, and the amino acid sequence of the signal
peptide of SlpA (CspA) is shown in SEQ ID NO: 6. In addition,
according to U.S. Pat. No. 4,965,197, it is said that a coryneform
bacterium-derived DNase also has a signal peptide, and such a
signal peptide can also be utilized in the present invention.
[0211] Signal peptides have constant, common features in the
sequence among biological species. However, a signal peptide which
exhibits a secretory function in one species does not necessarily
exhibit a secretory function in another species. Thus, in a case
where a heterologous signal peptide is used, one which functions in
the host to be used should be selected as appropriate. Whether or
not a certain signal peptide functions in the host to be used can
be confirmed, for example, by expressing the target protein fused
to the signal peptide to test whether or not the protein is
secreted.
[0212] A signal sequence is generally cleaved by a signal peptidase
when the translated product is secreted outside the microbial
cells. The signal peptidase used may be one that the host to be
used inherently has, or a gene encoding the signal peptidase which
functions in the host may be incorporated into the host.
[0213] The gene encoding the signal peptide can be used in a
naturally-occurring form, or may be modified so as to have optimum
codons in accordance with the codon usage in the host to be
used.
[0214] In the case where the protein having a self-assembly
capability is located upstream of the target protein, a gene
construct for expressing and secreting the fusion protein has the
nucleic acid sequence (added sequence) encoding the protein having
a self-assembly capability inserted between the nucleic acid
sequence encoding the signal peptide and the nucleic acid sequence
encoding the target protein. In the case where the protein having a
self-assembly capability is located downstream of the target
protein, the nucleic acid sequence (added sequence) encoding the
protein having a self-assembly capability is inserted downstream of
the nucleic acid sequence encoding the target protein.
[0215] It should be noted that a nucleic acid sequence encoding an
amino acid sequence used for enzymatic or chemical cleavage may be
further incorporated between the nucleic acid sequence encoding the
added sequence and the nucleic acid sequence encoding the target
protein. By inserting the amino acid sequence used for enzymatic or
chemical cleavage into the fusion protein, the expressed fusion
protein can be enzymatically or chemically cleaved to obtain the
target protein.
[0216] The cleaving can be performed enzymatically or chemically
according to conventional methods.
[0217] The cleaving step can be performed simultaneously with step
(4), during step (4), or after step (4).
[0218] The phrase "simultaneously with step (4)" means that step
(4) and the cleaving step are simultaneously performed using a
solution obtained by adding in advance a cleaving reagent (for
example, a protease used for an enzymatic cleavage to be described
later) to the "solution having a pH of 12 or below but higher than
the pH of the solution obtained in step (2) by 0.1 or more" used in
step (4).
[0219] The phrase "during step (4)" means that step (4) is
initiated without adding the cleaving reagent to the "solution
having a pH of 12 or below but higher than the pH of the solution
obtained in step (2) by 0.1 or more" used in step (4), and then the
reagent is added during step (4) to perform the cleaving step.
[0220] The phrase "after step (4)" means that after step (4) is
completed, the cleaving step is performed using the cleaving
reagent.
[0221] After the cleaving step, the target protein can be easily
separated from the protein having a self-assembly capability by
adopting methods well-known and commonly-used in this technical
field. In this event, only the protein having a self-assembly
capability may be precipitated by adjusting a pH of the solution
having been subjected to the cleaving step in the same manner as in
step (2), and only a solid of the protein having a self-assembly
capability may be separated in the same manner as in step (3).
[0222] The amino acid sequence used for the enzymatic cleavage is
not particularly limited, as long as the sequence is recognized and
cleaved by an enzyme capable of causing hydrolysis of a peptide
bond. A sequence usable in accordance with the amino acid sequence
of the target protein should be selected as appropriate. A nucleic
acid sequence encoding the amino acid sequence used for the
enzymatic cleavage should be designed as appropriate based on the
amino acid sequence. Moreover, optimum codons should be used in
accordance with the codon usage in the host, for example.
[0223] The amino acid sequence used for the enzymatic cleavage is
preferably a recognition sequence of a protease with a high
substrate specificity. Specific examples of such an amino acid
sequence include a Factor Xa protease recognition sequence, a
proTEV protease recognition sequence, and a trypsin recognition
sequence. A Factor Xa protease recognizes the amino acid sequence
of Ile-Glu-Gly-Arg (=IEGR) (SEQ ID NO: 7) in the protein, whereas a
ProTEV protease recognizes the amino acid sequence of
Glu-Asn-Leu-Tyr-Phe-Gln (=ENLYFQ) (SEQ ID NO: 8) in the protein.
Both of the proteases specifically cleave C-terminal sides of the
sequences. Trypsin recognizes Lys and Arg in the protein, and
specifically cleaves C-terminal sides of Lys and Arg.
[0224] The amino acid sequence used for the chemical cleavage is
not particularly limited, as long as the sequence is cleaved under
adopted chemical reaction conditions. A sequence usable in
accordance with the amino acid sequence of the target protein
should be selected as appropriate. A nucleic acid sequence encoding
the amino acid sequence used for the chemical cleavage should be
designed as appropriate based on the amino acid sequence. Moreover,
optimum codons should be used in accordance with the codon usage in
the host, for example.
[0225] The amino acid sequence used for the chemical cleavage is
preferably a sequence in which the cleaving occurs with a high
specificity. A specific example of such a cleavage site of the
amino acid sequence includes Met. If a cyanogen bromide degradation
method is employed, the cleaving occurs on the C-terminal side of
Met.
[0226] The "gene construct for expressing the fusion protein" can
be constructed according to techniques well-known and commonly-used
in this technical field.
[0227] The technique for introducing the gene construct into the
host is not particularly limited. It is possible to use
generally-used techniques, for example, a protoplast method (Gene,
39, 281-286 (1985)), an electroporation method (Bio/Technology, 7,
1067-1070) (1989)), and the like.
[0228] In a case where the host is a bacterium, the gene construct
may be present on a vector capable of self-replicating
extrachromosomally like a plasmid, or may be incorporated into a
chromosome.
[0229] For example, in a case where the gene construct is
introduced into the host coryneform bacterium using a vector, the
vector is not particularly limited, as long as it is capable of
self-replicating in the coryneform bacterium. The vector may be,
for example, vectors derived from bacterial plasmids, vectors
derived from yeast plasmids, vectors derived from bacteriophages,
cosmids, phagemids, or the like. The vector is preferably plasmids
derived from coryneform bacteria. The vector capable of
self-replicating in the coryneform bacterium specifically includes
pHM1519 (Agric, Biol. Chem., 48, 2901-2903 (1984)); pAM330 (Agric.
Biol. Chem., 48, 2901-2903 (1984)); plasmids improved from them to
have a drug resistant gene; a plasmid pCRY30 described in JP-A Hei
3-210184; plasmids pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE, and
pCRY3KX described in JP-A Hei 2-72876 and U.S. Pat. No. 5,185,262;
plasmids pCRY2 and pCRY3 described in JP-A Hei 1-191686; pAJ655,
pAJ611, and pAJ1844 described in JP-A Sho 58-192900; pCG1 described
in JP-A Sho 57-134500; pCG2 described in JP-A Sho 58-35197; pCG4
and pCG11 described in JP-A Sho 57-183799; and the like.
[0230] In introducing the gene construct, an artificial transposon
or the like can also be utilized. In a case of using a transposon,
the gene construct is introduced into a chromosome by homologous
recombination or by the transposition ability of the transposon
itself. Besides, examples of the introduction method utilizing
homologous recombination include methods using a linear DNA, a
plasmid containing a temperature sensitive replication origin, a
plasmid capable of conjugative transfer, a suicide vector not
having a replication origin which functions in the host, or the
like. Note that when the fusion protein gene is introduced into the
chromosome, one or more sequences contained in the gene construct,
which are selected from the promoter sequence, the nucleic acid
sequence encoding the signal peptide, and the nucleic acid sequence
encoding the added sequence, may be ones inherently present on the
chromosome of the host, as long as the gene construct is
constructed on the chromosome. For example, the gene construct can
be constructed on the chromosome as follows. Specifically, a
promoter sequence and a nucleic acid sequence encoding the signal
peptide, connected downstream of the promoter sequence, which are
inherently present on the chromosome of the host, are used as they
are; meanwhile, only the gene connected downstream of the nucleic
acid sequence encoding the signal peptide is replaced with the
nucleic acid sequence encoding the fusion protein made of the added
sequence and the target protein.
[0231] In a case where two or more types of the target protein are
produced, the host should contain gene constructs for expressing
the proteins so as to express the proteins. For example, all of the
gene constructs for expressing the proteins may be contained on a
single expression vector, or all of the gene constructs may be
contained on the chromosome. Alternatively, the gene constructs for
expressing the proteins may be contained separately on multiple
expression vectors, or may be contained separately on a single or
multiple expression vectors and on the chromosome. Incidentally,
the two or more types of the target protein include a
heteromultimeric protein.
[0232] By culturing the host having the gene construct(s)
introduced therein, and expressing the fusion protein to be
secreted outside the microbial cells or accumulated in the
microbial cells, a culture solution containing the fusion protein
is prepared.
[0233] The host can be cultured according to normally-used methods
and conditions. For example, when a bacterium is used as the host,
the bacterium can be cultured in a normal medium containing a
carbon source, a nitrogen source, and inorganic ions. In order to
achieve a higher growth, organic trace nutrients such as vitamins
and amino acids may also be added as necessary.
[0234] As the carbon source, it is possible to use carbohydrates
such as glucose and sucrose, organic acids such as acetic acid,
alcohols, or others. As the nitrogen source, it is possible to use
an ammonia gas, ammonia water, ammonium salts, or others. As the
inorganic ions, it is possible to use calcium ions, magnesium ions,
phosphate ions, potassium ions, iron ions, or the like as
appropriate if necessary.
[0235] The culturing can be performed under appropriate conditions
for the host to be used. For example, in a case of a coryneform
bacterium, the culturing is performed within suitable ranges of pH
5.0 to 8.5 and 15.degree. C. to 37.degree. C. under aerobic
conditions for approximately 1 to 7 days. Moreover, it is possible
to adopt culture conditions for producing an L-amino acid of
coryneform bacteria, or conditions described in the method for
producing a protein using a signal peptide of the Sec system or Tat
system (see WO01/23591, WO2005/103278).
[0236] In a case where an inducible promoter is used to express the
fusion protein, the culturing can be performed by adding a promoter
inducer to the medium.
[0237] Whether the fusion protein is produced or not can be
confirmed from the molecular weight of a protein band separated in
SDS-PAGE performed on a sample of fractions containing a culture
supernatant (including cell homogenate also in a case where the
fusion protein is accumulated in the microbial cells). Moreover, it
can be confirmed by western blot using an antibody, which is
performed on a sample of fractions containing a culture supernatant
(including the above-described cell homogenate also) (Molecular
cloning (Cold spring Harbor Laboratory Press, Cold spring Harbor
(USA), 2001)). Further, it can be confirmed by determining the
N-terminal amino acid sequence of the produced protein using a
protein sequencer. Furthermore, it can be confirmed also by
determining the mass of the produced protein using a mass
spectrometer.
[0238] Additionally, in some cases, the concentration and purity of
the fusion protein (and also the target protein) can also be
determined using reversed-phase HPLC.
[0239] When the fusion protein is secreted outside the host
microbial cells, a "solution containing the fusion protein" may be
the above-described culture solution containing the fusion protein
itself (culture solution containing the microbial cells), or may be
a culture supernatant obtained by separating the host microbial
cells from the culture solution (microorganism-removed culture
solution). Specifically, the "solution containing the fusion
protein" (hereinafter may also be referred to as "solution obtained
in step (1)") includes not only the culture supernatant obtained by
removing the microbial cells from the culture solution containing
the fusion protein, but also the culture solution itself containing
the fusion protein. In the present invention, the culture
supernatant is preferably used.
[0240] The microbial cells can be separated from the culture
solution by methods well-known and commonly-used in this technical
field, for example, centrifugation, membrane filtration, and the
like. The step of separating the microbial cells is normally
performed in step (1) from the viewpoint of improving the purity
and the recovery ratio of the fusion protein (or the target
protein). Nevertheless, when it is preferable to perform the
separation in another step for various reasons, the separation may
be performed in any step other than step (1).
[0241] When the fusion protein is accumulated in the form of a
soluble protein in the host microbial cells, the "solution obtained
in step (1)" can be prepared by disrupting the microbial cells, and
separating solid matters from the homogenate. The microbial cells
can be disrupted according to methods well-known and commonly-used
in this technical field, for example, a homogenation method.
[0242] When the fusion protein is accumulated in the form of the
insoluble protein in the host microbial cells, the "solution
obtained in step (1)" can be prepared by disrupting the microbial
cells, collecting solid matters from the homogenate, and subjecting
the solid matters to well-known and commonly-used methods, for
example, a solubilization treatment using a protein denaturing
agent such as urea and guanidine or a surfactant such as SDS, to
then separate the solid matters. The microbial cells can be
disrupted by methods well-known and commonly-used in this technical
field, for example, a homogenation method.
[0243] In step (2) of the production method of the present
invention, a pH of the solution obtained in step (1) is adjusted to
such a pH that a recovery ratio calculated according to the
following equation is 100 or more, where
the recovery ratio (%)=[an amount of the fusion protein in a
solution obtained in step (4)/{the amount of the fusion protein in
the solution obtained in step (4)+an amount of the fusion protein
in a solution after solid separation in step (3)}].times.100.
[0244] It should be noted that the terms related to "precipitation"
used in the description hereinafter (for example, "precipitate",
"precipitate-dissolved solution," and the like) are directed to the
fusion protein, unless otherwise specifically stated. In a case
where "precipitation" is directed to the host microbial cells, such
an explanation will be given.
[0245] In addition, the description related to the analysis of the
fusion protein or the target protein is directed to samples from
which the microbial cells have been removed.
[0246] "The amount of the fusion protein in the solution obtained
in step (4)" and "the amount of the fusion protein in the solution
after solid separation in step (3)" in the equation for the
recovery ratio can be determined using protein quantification
methods well-known and commonly-used in this technical field, for
example, reducing SDS-PAGE, reversed-phase HPLC, and the like.
[0247] The quantification of the fusion protein by reducing
SDS-PAGE can be performed using methods well-known and
commonly-used in this technical field, for example, using as an
indicator the band intensity of the fusion protein after reducing
SDS-PAGE described in Example 1. In this case, the equation for the
recovery ratio is as follows:
the recovery ratio (%)=[a band intensity of the fusion protein in
the solution obtained in step (4)/{the band intensity of the fusion
protein in the solution obtained in step (4)+a band intensity of
the fusion protein in the solution after solid separation in step
(3)}].times.100.
[0248] The quantification of the fusion protein by reversed-phase
HPLC can be performed using methods well-known and commonly-used in
this technical field, for example, a quantification method based on
a peak area of the fusion protein in a chromatogram obtained by
reversed-phase HPLC described in Example 1-2.
[0249] The recovery ratio may vary, depending on the type and usage
of the target protein, the necessary amount, and the like. However,
the recovery ratio is 10% or more, preferably 20% or more, and
further preferably 30% or more.
[0250] Note that the upper limit of the pH to achieve the recovery
ratio of 10% or more can be easily determined, as described in
Example 1 later, by: conducting multiple tests (each test includes
steps (1) to (4)) at various pHs with which step (2) is performed;
calculating the recovery ratio in each test; and creating a
relation graph between the calculated recovery ratio and the
pH.
[0251] The pH value determined based on the above equations is the
upper limit of the pH applicable in step (2) (pH at which the
recovery ratio becomes 10% or more). Thus, step (2) can be
performed with a pH equal to or below the upper limit of the pH
determined above. Meanwhile, the lower limit of the pH is not
particularly limited, as long as the fusion protein is not
irreversibly denatured by acid. The lower limit of the pH is
determined in consideration of the cost for the pH adjustment
operation and a desired recovery ratio.
[0252] The pH value used in step (2) may vary, depending on the
type and usage of the target protein, the necessary amount, and the
like. However, the pH is preferably 9 or below. For example, the pH
is -0.5 to 9, preferably 1.5 to 9.
[0253] A substance used for the pH adjustment is not particularly
limited, and all types of acids and alkalis can be used. Examples
of the acids include sulfuric acid, hydrochloric acid, acetic acid,
phosphoric acid, trifluoroacetic acid (TFA), and the like. Among
these, sulfuric acid, hydrochloric acid, and acetic acid are
preferable. One acid may be used alone, or two or more acids may be
used in combination. Examples of the bases include sodium
hydroxide, ammonia, tris(hydroxymethyl)aminomethane (Tris), and the
like. One base may be used alone, or two or more bases may be used
in combination.
[0254] In step (2), after the pH is adjusted, a solid is formed in
the solution. The solid is mainly formed of the fusion protein.
This fusion protein is not substantially denatured by acid. In
order to facilitate the solid formation, the solution is preferably
stirred or left alone after the pH adjustment.
[0255] Incidentally, in a case where the solution obtained in step
(1) already has a pH value that should be achieved by the pH
adjustment operation in step (2), the pH adjustment operation in
step (2) is not necessary, and step (3) can be performed after the
solution obtained in step (1) is stirred or left alone as
appropriate.
[0256] Thus, as an embodiment, the method for producing a fusion
protein of the present invention comprises the following steps (A)
to (C):
[0257] (A) preparing a solution containing the fusion protein, the
solution having such a pH that a recovery ratio calculated
according to the following equation is 10% or more, where
the recovery ratio (%)=[an amount of the fusion protein in a
solution obtained in step (C)/{the amount of the fusion protein in
the solution obtained in step (C)+an amount of the fusion protein
in a solution after solid separation in step (B)}].times.100;
[0258] (B) separating a solid from the solution obtained in step
(A); and
[0259] (C) dissolving the solid separated in step (B) into a
solution having a pH of 12 or below but higher than the pH of the
solution obtained in step (A) by 0.1 or more.
[0260] Note that the above descriptions related to steps (1) and
(2) can apply to step (A). Moreover, descriptions related to steps
(3) and (4) below can apply to steps (B) and (C).
[0261] In step (3) of the production method of the present
invention, the solid is separated from the solution obtained in
step (2). In the solution obtained in step (2), various components
(for example, colored substances, lipids, and impurity proteins)
derived from the microbial cells and components (for example,
colored substances and inorganic salts) derived from the medium
remain other than the fusion protein. These can be removed by step
(3), consequently making it possible to easily improve the purity
of the fusion protein.
[0262] The separation can be performed by methods well-known and
commonly-used in this technical field. For example, centrifugation,
membrane filtration, and the like can be used. Multiple methods may
be performed in combination (for example, a combination of
centrifugation and membrane filtration).
[0263] The solid obtained in step (3) may have a liquid
(impurities) not having been removed completely and attached to the
solid. When a solution having a pH equal to or below the upper
limit of the pH applicable in step (2) (i.e., the pH at which the
recovery ratio of the fusion protein becomes 10%) is added to the
solid having the impurities attached thereto, a mixture
(suspension) of the solid with the solution (including the liquid
(impurities)) is obtained. By separating the solid from the
suspension again, a solid having a smaller amount of the impurities
attached thereto can be obtained. After step (3), this operation
may be performed once or several times.
[0264] In step (4) of the production method of the present
invention, the solid separated in step (3) is dissolved into the
solution having a pH of 12 or below but higher than the pH of the
solution obtained in step (2) by 0.1 or more.
[0265] A more preferable pH of the solution used in step (4) is
higher than the pH at which the recovery ratio of the targeted
fusion protein is 10% (i.e., the upper limit of the pH applicable
in step (2)) by 0.1 or more, more preferably 0.2 or more, and
further preferably 1 or more. The use of the preferable pH in this
range makes it possible to sufficiently dissolve the fusion protein
into the solution.
[0266] The preferable pH may vary, depending on the type of the
protein having a self-assembly capability and the type of the
target protein. Nevertheless, for example, in a case where the
protein having a self-assembly capability is the CspB mature
protein or a portion thereof, the pH is preferably 12 or below,
more preferably 11 or below, and further preferably 10 or
below.
[0267] The solution for dissolving the solid separated in step (3)
is not particularly limited and any type of substance can be used,
as long as the solution has the above-described pH. A specific
example thereof includes a buffer. A buffer is preferable because
the pH is less likely to change. The buffer includes ones obtained
by using Tris, HEPES, sodium phosphate, citric acid, or the like,
and Tris is preferable.
[0268] By performing step (4), the fusion protein can be obtained
in the form of solution. Increasing or decreasing the amount of the
solution used in this step can adjust the concentration of the
fusion protein as needed. Moreover, the composition of the solution
used in step (4) can be determined in consideration of step
performed next. Thus, performing steps (2) to (4) makes it possible
to easily improve the purity of the fusion protein, and/or
concentrate the fusion protein solution, and/or replace the
solvent.
[0269] Note that when the solid is formed in step (2), substances
other than the fusion protein are included as impurities in the
solid, and stay remained in the solution obtained in step (4) in
some cases. In such a case, by performing steps (2) to (4) again
using the solution obtained in step (4) as the solution obtained in
step (1), the amount of the impurities can be reduced. The number
of times of the operation to be performed after step (4) may be one
or more.
[0270] In the solution obtained in step (4), the fusion protein is
dissolved.
[0271] By cleaving the protein having a self-assembly capability
from the fusion protein, the target protein can be obtained.
[0272] The cleaving can be performed according to methods
well-known and commonly-used in this technical field. Examples of
the cleaving method include an enzymatic cleavage, a chemical
cleavage, and the like. An enzymatic cleavage is preferable because
the cleaving specificity is excellent. The cleaving step may be
performed on the solution containing the fusion protein obtained in
step (4), or may be performed after the fusion protein is separated
from the solution obtained in step (4). In the case where the step
is performed on the solution containing the fusion protein obtained
in step (4), the cleaving may be performed simultaneously with step
(4), during step (4), or after step (4).
[0273] The enzymatic cleavage can be suitably performed by
incorporating a recognition sequence of a protease with a high
substrate specificity (for example, the aforementioned Factor Xa
protease recognition sequence, proTEV protease recognition
sequence, trypsin recognition sequence) between the protein having
a self-assembly capability and the target protein.
[0274] The fusion protein obtained in step (4) or the target
protein obtained by cleaving the fusion protein can be purified
from the solution according to methods well-known and commonly-used
in this technical field. The fusion protein or the target protein
can be purified by subjecting the solution containing the fusion
protein or the target protein to a suitable known method such as,
for example, column chromatography (for example, high-performance
liquid chromatography (HPLC), reversed-phase chromatography (for
example, reversed-phase HPLC), medium high-pressure liquid
chromatography, ion-exchange column chromatography, hydrophobic
chromatography, gel filtration chromatography, affinity
chromatography), alcohol precipitation, or ultrafiltration, or a
combination of these.
[0275] Note that when the solution obtained after the step of
cleaving the fusion protein is subjected to the same step as step
(2), the protein having a self-assembly capability is precipitated.
By performing this step, the target protein can be purified more
efficiently.
[0276] Whether or not the target protein is obtained can be
confirmed according to analysis methods well-known and
commonly-used in this technical field. For example, it can be
confirmed by subjecting a sample to SDS-PAGE, and checking the
molecular weight of a protein band thus separated. Moreover, it can
be confirmed by western blot using an antibody (Molecular cloning
(Cold spring Harbor Laboratory Press, Cold spring Harbor (USA),
2001)). Further, it can also be confirmed by determining the
N-terminal amino acid sequence of the target protein using a
protein sequencer. Furthermore, it can also be confirmed by
determining the mass of the target protein using a mass
spectrometer. Additionally, in a case where the target protein has
enzymatic or certain measureable physiological activity, the
confirmation is possible also using the enzymatic activity or
physiological activity as an indicator.
[0277] The target protein obtained according to the present
invention may be used as it is, or may be further modified. The
modification includes amino acid addition by chemical synthesis,
PEGylation (Biotechnol J., January; 5 (1): 113 (2010)), and the
like. For example, in a case where Biva18 (peptide obtained by
deleting 2 amino acid residues (D-Phe-L-Pro) from the N-terminus of
a bioactive protein bivalirudin (20 amino acids)) is produced as
the target protein, full length bivalirudin having a physiological
activity can be prepared by adding 2 amino acid residues
(D-Phe-L-Pro) to the N-terminus of Biva18 by chemical synthesis.
Specifically, full length bivalirudin can be prepared by a chemical
synthesis reaction by which particular activated amino acid
residues react with the N-terminus of Biva18.
[0278] The modified target protein can be purified by methods
well-known and commonly-used in this technical field, for example,
ion-exchange chromatography, reversed-phase HPLC, hydrophobic
chromatography, and the like.
EXAMPLES
[0279] Hereinafter, the present invention will be further
specifically described based on Examples and Comparative Example.
Nevertheless, the present invention is not limited to these
Examples.
Example 1
Production of Fusion Protein Having Bioactive Peptide
Teriparatide
[0280] In Example 1, a bioactive peptide teriparatide (34 amino
acid residues) (Teri) was used as a target protein, a sequence
consisting of 50 amino acid residues from the N-terminus of a CspB
mature protein (CspB50) was used as a protein having a
self-assembly capability, a proTEV protease recognition sequence
was used as an amino acid sequence used for an enzymatic cleavage,
and a coryneform bacterium C. glutamicum was used as a host.
(1) Construction of Teriparatide-Secretory Expression Plasmid
pPKK50TEV-Teri (i) Total Synthesis of Proinsulin Gene, and
Construction of Proinsulin-Secretory Expression Plasmid pPKPIns
Using Signal Sequence of Cell Surface Protein CspB of C. glutamicum
ATCC13869
[0281] The amino acid sequence of proinsulin (hereinafter described
as PIns) has already been determined (Genbank Accession No.
NP.sub.--000198.1). In consideration of this sequence and the codon
usage of C. glutamicum, DNAs shown in <SEQ ID NO: 9> to
<SEQ ID NO: 16> were synthesized. Using these DNAs as
templates, and using DNAs shown in <SEQ ID NO: 17> and
<SEQ ID NO: 18> as primers, a gene encoding PIns was
amplified by PCR. Thus, approximately 0.3 kbp of a DNA fragment
shown in <SEQ ID NO: 19> was obtained. This DNA fragment was
inserted in a SmaI site of a cloning vector pHSG398 (manufactured
by Takara Bio Inc.) to thus obtain pHSG-PIns. Using this pHSG-PIns
as a template, and using the DNAs shown in <SEQ ID NO: 17>
and <SEQ ID NO: 18> as primers, a PIns gene region was
amplified by PCR. Thus, approximately 0.3 kbp of a PIns gene
fragment was obtained.
[0282] On the other hand, the base sequence of a gene encoding the
cell surface protein CspB of C. glutamicum has already been
determined (Mol. Microbiol., 9, 97-109 (1993)). Referring to this
sequence, pPKPTG1 described in WO01/23591 (the pPKPTG1 is a
protransglutaminase (transglutaminase having a pro-structural
portion)-secretory expression vector having: a promoter of a cspB
gene derived from a C. glutamicum ATCC13869 strain; a DNA encoding
30 amino acid residues of a signal peptide of CspB derived from the
C. glutamicum ATCC13869 strain, the DNA expressibly ligated
downstream of the promoter; and a protransglutaminase gene derived
from an actinobacterium Streptoverticillium mobaraense, the gene
ligated downstream of the DNA encoding the signal peptide in such a
manner as to be expressed in the form of a fusion protein with the
signal peptide) was used as a template, and primers shown in
<SEQ ID NO: 20> and <SEQ ID NO: 21> were used in PCR to
amplify a region encoding the promoter region of CspB derived from
the C. glutamicum ATCC13869 strain and the signal peptide. Thus,
approximately 0.7 kbp of a DNA fragment was obtained.
[0283] Then, using the two amplified DNA fragments (the PIns gene
fragment; the fragment of the region encoding the promoter region
and the signal peptide) as templates, and using the DNAs shown in
<SEQ ID NO: 20> and <SEQ ID NO: 18> as primers,
approximately 0.9 kbp of a DNA fragment was obtained by PCR, in
which the two DNA fragments were fused together. Note that the
primers of <SEQ ID NO: 20> and <SEQ ID NO: 18> were
each designed to have a restriction enzyme KpnI recognition
sequence. In the PCR reaction, Pyrobest DNA polymerase
(manufactured by Takara Bio Inc.) was used, and the reaction
conditions were in accordance with the manufacturer's recommended
protocol. After restriction enzyme KpnI treatment, the DNA fragment
was inserted in a KpnI site of pPK4 described in JP-A Hei 9-322774
to thus obtain pPKPIns.
[0284] The result of determining the base sequence of the inserted
fragment confirmed that the fusion gene was constructed as
expected. Note that the base sequence was determined using
BigDye.RTM. Terminator v3.1 Cycle Sequencing Kit (manufactured by
Applied BioSystems Inc.) and 3130 Genetic Analyzer (manufactured by
Applied BioSystems Inc.).
(ii) Construction of Secretory Expression Plasmids for Proinsulin
Fused with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17,
20, 50, 100, 150, 200, 250, 300, 350, 400, or 440 Amino acid
residues at N-Terminus of Mature Cell Surface Protein CspB of C.
glutamicum ATCC13869
[0285] As described above, the base sequence of the gene encoding
the cell surface protein CspB of C. glutamicum has already been
determined (Mol. Microbiol., 9, 97-109 (1993)). It is known that
CspB is localized in the cell surface layer of C. glutamicum,
forming a layer called an S-layer, and a highly hydrophobic amino
acid residue region on the C-terminal side is involved in the
localization (Mol. Microbiol., 9, 97-109 (1993)). Referring to the
sequence, primers shown in <SEQ ID NO: 20> and <SEQ ID NO:
22> were synthesized, and using a chromosomal DNA of C.
glutamicum ATCC13869 as a template prepared in accordance with an
ordinary method (the method by Saito and Miura [Biochem. Biophys.
Act., 72, 619 (1963)]), a region encoding a 5'-upstream region
containing the promoter of the gene encoding CspB (hereinafter may
also be referred to as CspB promoter region), the 30 amino acid
residues of the signal peptide at the N-terminus of CspB, and 440
amino acid residues at the N-terminus of the CspB mature protein
was amplified by PCR. Note that the 440 amino acid residues at the
N-terminus of the CspB mature protein were obtained from the full
length 469 amino acids of the CspB mature protein of C. glutamicum
ATCC13869 (SEQ ID NO: 3) by excluding 29 amino acids in the
hydrophobic region on the C-terminal side therefrom. In the PCR
reaction, Pyrobest DNA polymerase (manufactured by Takara Bio Inc.)
was used, and the reaction conditions were in accordance with the
manufacturer's recommended protocol.
[0286] Next, in order to construct secretory expression plasmids
for proinsulin fused with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 17, 20, 50, 100, 150, 200, 250, 300, 350, 400, or 440
amino acid residues at the N-terminus of the mature cell surface
protein CspB of C. glutamicum, regions encoding the CspB promoter
region, the 30 amino acid residues of the signal peptide at the
N-terminus of CspB, and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 17, 20, 50, 100, 150, 200, 250, 300, 350, 400, or 440 amino
acid residues at the N-terminus of the CspB mature protein were
amplified by PCR using the above-amplified PCR reaction product as
a template, and using corresponding synthetic DNAs shown in <SEQ
ID NO: 20> and <SEQ ID NO: 23>, <SEQ ID NO: 20> and
<SEQ ID NO: 24>, <SEQ ID NO: 20> and <SEQ ID NO:
25>, <SEQ ID NO: 20> and <SEQ ID NO: 26>, <SEQ ID
NO: 20> and <SEQ ID NO: 27>, <SEQ ID NO: 20> and
<SEQ ID NO: 28>, <SEQ ID NO: 20> and <SEQ ID NO:
29>, <SEQ ID NO: 20> and <SEQ ID NO: 30>, <SEQ ID
NO: 20> and <SEQ ID NO: 31>, <SEQ ID NO: 20> and
<SEQ ID NO: 32>, <SEQ ID NO: 20> and <SEQ ID NO:
33>, <SEQ ID NO: 20> and <SEQ ID NO: 34>, <SEQ ID
NO: 20> and <SEQ ID NO: 35>, <SEQ ID NO: 20> and
<SEQ ID NO: 36>, <SEQ ID NO: 20> and <SEQ ID NO:
37>, <SEQ ID NO: 20> and <SEQ ID NO: 38>, <SEQ ID
NO: 20> and <SEQ ID NO: 39>, <SEQ ID NO: 20> and
<SEQ ID NO: 40>, <SEQ ID NO: 20> and <SEQ ID NO:
41>, <SEQ ID NO: 20> and <SEQ ID NO: 42>, <SEQ ID
NO: 20> and <SEQ ID NO: 43>, <SEQ ID NO: 20> and
<SEQ ID NO: 44>, <SEQ ID NO: 20> and <SEQ ID NO:
45>, <SEQ ID NO: 20> and <SEQ ID NO: 46>, <SEQ ID
NO: 20> and <SEQ ID NO: 47>, or <SEQ ID NO: 20> and
<SEQ ID NO: 48> as primers. On the other hand, using the
plasmid pPKPIns constructed above in (i) as a template, and using
synthetic DNAs shown in <SEQ ID NO: 17> and <SEQ ID NO:
49> as primers, a PIns gene region was amplified by PCR. Thus, a
PIns gene fragment was obtained.
[0287] Then, using the two amplified DNA fragments (the fragment of
the region encoding the CspB promoter region, the CspB signal
peptide, and the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
17, 20, 50, 100, 150, 200, 250, 300, 350, 400, or 440 amino acid
residues at the N-terminus of the mature CspB; the PIns gene
fragment) as templates, and using the DNAs shown in <SEQ ID NO:
20> and <SEQ ID NO: 49> as primers, DNA fragments were
obtained by PCR, in each of which the two DNA fragments were fused
together. Note that the primers of <SEQ ID NO: 20> and
<SEQ ID NO: 49> were each designed to have the restriction
enzyme KpnI recognition sequence, and the primers of <SEQ ID NO:
23> to <SEQ ID NO: 48> each contained a sequence encoding
an amino acid sequence on the N-terminal side of PIns so as to
construct a fusion gene made of the PIns gene and the region
encoding the N-terminus of the CspB mature protein. In the PCR
reaction, Pyrobest DNA polymerase (manufactured by Takara Bio Inc.)
was used, and the reaction conditions were in accordance with the
manufacturer's recommended protocol. After restriction enzyme KpnI
treatment, each of these DNA fragments was inserted in a KpnI site
of pPK4 described in JP-A Hei 9-322774 to thus obtain pPKK1PIns,
pPKK2PIns, pPKK3PIns, pPKK4PIns, pPKK5PIns, pPKK6PIns, pPKK7PIns,
pPKK8PIns, pPKK9PIns, pPKK10PIns, pPKK11PIns, pPKK12PIns,
pPKK13PIns, pPKK14PIns, pPKK15PIns, pPKK17PIns, pPKK20PIns,
pPKK50PIns, pPKK100PIns, pPKK150PIns, pPKK200PIns, pPKK250PIns,
pPKK300PIns, pPKK350PIns, pPKK400PIns, and pPKK440PIns. The result
of determining the base sequences of the inserted fragments
confirmed that the fusion genes were constructed as expected. Note
that the base sequences were determined using BigDye.RTM.
Terminator v3.1 Cycle Sequencing Kit (manufactured by Applied
BioSystems Inc.) and 3130 Genetic Analyzer (manufactured by Applied
BioSystems Inc.).
(iii) Construction of Plasmids pPKK17Xa-PIns and pPKK50Xa-PIns
[0288] Using pPKK17PIns or pPKK50PIns constructed above in (ii) as
a template, and using corresponding synthetic DNAs shown in <SEQ
ID NO: 20> and <SEQ ID NO: 50> or <SEQ ID NO: 20>
and <SEQ ID NO: 51> as primers, fragments were amplified by
PCR, in each of which a region encoding IEGR recognized by a Factor
Xa protease was further added to the region encoding the CspB
promoter region, the 30 amino acid residues of the signal peptide
at the N-terminus of CspB, and the 17 or 50 amino acid residues at
the N-terminus of the CspB mature protein. On the other hand, using
the plasmid pPKPIns constructed above in (i) as a template, and
using corresponding synthetic DNAs shown in <SEQ ID NO: 52>
and <SEQ ID NO: 49> or <SEQ ID NO: 53> and <SEQ ID
NO: 49> as primers, a PIns gene region was amplified by PCR.
Thus, a PIns gene fragment was obtained. Then, using the two
amplified DNA fragments (the fragment of the region encoding the
CspB promoter region, the CspB signal peptide, the 17 or 50 amino
acid residues at the N-terminus of the mature CspB (QETNPT), and
IEGR; the PIns gene fragment) as templates, and using the DNAs
shown in <SEQ ID NO: 20> and <SEQ ID NO: 49> as
primers, DNA fragments were obtained by PCR, in each of which the
two DNA fragments were fused together. Note that the primers of
<SEQ ID NO: 20> and <SEQ ID NO: 49> were each designed
to have the restriction enzyme KpnI recognition sequence, and the
primers of <SEQ ID NO: 50> and <SEQ ID NO: 51> were
each designed to have a sequence encoding an amino acid sequence on
the N-terminal side of PIns so as to construct a fusion gene made
of the PIns gene and the base sequence encoding IEGR. In the PCR
reaction, Pyrobest DNA polymerase (manufactured by Takara Bio Inc.)
was used, and the reaction conditions were in accordance with the
manufacturer's recommended protocol. After restriction enzyme KpnI
treatment, each of these DNA fragments was inserted in a KpnI site
of pPK4 described in JP-A Hei 9-322774 to thus obtain pPKK17Xa-PIns
and pPKK50Xa-PIns. The result of determining the base sequences of
the inserted fragments confirmed that the fusion genes were
constructed as expected. Note that the base sequences were
determined using BigDye.RTM. Terminator v3.1 Cycle Sequencing Kit
(manufactured by Applied BioSystems Inc.) and 3130 Genetic Analyzer
(manufactured by Applied BioSystems Inc.).
(iv) Construction of Secretory Expression Plasmids for Fusion
Proinsulin Having Factor Xa Protease or ProTEV Protease Recognition
Sequence Inserted between N-Terminal Amino Acid Sequence of CspB
Mature Protein and Proinsulin Sequence
[0289] For expressing a certain target protein in a form fused with
an amino acid sequence other than that of the target protein, there
is well known a method for obtaining the target protein easily by
disposing a particular protease recognition sequence of a protease
with a high substrate specificity between the amino acid sequence
of the target protein and the fused amino acid sequence to thereby
cleave the expressed fusion protein with the particular protease.
On the other hand, a Factor Xa protease, a ProTEV protease, and the
like are known as proteases with a high substrate specificity, and
respectively recognize sequences Ile-Glu-Gly-Arg (=IEGR) (SEQ ID
NO: 7) and Glu-Asn-Leu-Tyr-Phe-Gln (=ENLYFQ) (SEQ ID NO: 8) in a
protein, and specifically cleave C-terminal sides of the sequences.
Hence, for example, if fusion PIns is expressed and secreted from
CspB-fused PIns by constructing a fusion PIns gene having a base
sequence encoding a Factor Xa protease recognition sequence (IEGR)
or a ProTEV protease recognition sequence (ENLYFQ) inserted between
a base sequence encoding amino acid residues at the N-terminus of
the CspB mature protein and a base sequence encoding proinsulin,
PIns can be easily obtained from the fusion PIns using these
proteases.
[0290] Using pPKK6PIns constructed above in (ii) as a template, and
using corresponding synthetic DNAs shown in <SEQ ID NO: 20>
and <SEQ ID NO: 54> or <SEQ ID NO: 20> and <SEQ ID
NO: 55> as primers, fragments were amplified by PCR, in each of
which a region encoding IEGR recognized by the Factor Xa protease
or ENLYFQ recognized by the ProTEV protease was further added to
the region encoding the CspB promoter region, the 30 amino acid
residues of the signal peptide at the N-terminus of CspB, and the 6
amino acid residues at the N-terminus of the CspB mature protein
(QETNPT). On the other hand, using the plasmid pPKPIns constructed
above in (i) as a template, and using the corresponding synthetic
DNAs shown in <SEQ ID NO: 52> and <SEQ ID NO: 49> or
<SEQ ID NO: 53> and <SEQ ID NO: 49> as primers, a PIns
gene region was amplified by PCR. Thus, a PIns gene fragment was
obtained. Then, using the two amplified DNA fragments (the fragment
of the region encoding the CspB promoter region, the CspB signal
peptide, the 6 amino acid residues at the N-terminus of the mature
CspB (QETNPT), and IEGR or ENLYFQ; the PIns gene fragment) as
templates, and using the DNAs shown in <SEQ ID NO: 20> and
<SEQ ID NO: 49> as primers, DNA fragments were obtained by
PCR, in each of which the two DNA fragments were fused together.
Note that the primers of <SEQ ID NO: 20> and <SEQ ID NO:
49> were each designed to have the restriction enzyme KpnI
recognition sequence, the primer of <SEQ ID NO: 54> was
designed to have a sequence encoding an amino acid sequence on the
N-terminal side of PIns so as to construct a fusion gene made of
the PIns gene and the base sequence encoding IEGR, and the primer
of <SEQ ID NO: 55> was designed to have a sequence encoding
an amino acid sequence on the N-terminal side of PIns so as to
construct a fusion gene made of the PIns gene and the base sequence
encoding ENLYFQ. In the PCR reaction, Pyrobest DNA polymerase
(manufactured by Takara Bio Inc.) was used, and the reaction
conditions were in accordance with the manufacturer's recommended
protocol. After restriction enzyme KpnI treatment, each of these
DNA fragments was inserted in a KpnI site of pPK4 described in JP-A
Hei 9-322774 to thus obtain pPKK6Xa-PIns and pPKK6TEV-PIns.
[0291] Similarly, using pPKK17PIns or pPKK50PIns constructed above
in (ii) as a template, and using the corresponding synthetic DNAs
shown in <SEQ ID NO: 20> and <SEQ ID NO: 50> or <SEQ
ID NO: 20> and <SEQ ID NO: 51> as primers, fragments were
amplified by PCR, in each of which the region encoding IEGR
recognized by the Factor Xa protease was further added to the
region encoding the CspB promoter region, the 30 amino acid
residues of the signal peptide at the N-terminus of CspB, and the
17 or 50 amino acid residues at the N-terminus of the CspB mature
protein. On the other hand, using the plasmid pPKPIns constructed
above in (i) as a template, and using the corresponding synthetic
DNAs shown in <SEQ ID NO: 52> and <SEQ ID NO: 49> or
<SEQ ID NO: 53> and <SEQ ID NO: 49> as primers, a PIns
gene region was amplified by PCR. Thus, a PIns gene fragment was
obtained. Then, using the two amplified DNA fragments (the fragment
of the region encoding the CspB promoter region, the CspB signal
peptide, the 17 or 50 amino acid residues at the N-terminus of the
mature CspB (QETNPT), and IEGR; the PIns gene fragment) as
templates, and using the DNAs shown in <SEQ ID NO: 20> and
<SEQ ID NO: 49> as primers, DNA fragments were obtained by
PCR, in each of which the two DNA fragments were fused together.
Note that the primers of <SEQ ID NO: 20> and <SEQ ID NO:
49> were each designed to have the restriction enzyme KpnI
recognition sequence, and the primers of <SEQ ID NO: 50> and
<SEQ ID NO: 51> were each designed to have the sequence
encoding the amino acid sequence on the N-terminal side of PIns so
as to construct the fusion gene made of the PIns gene and the base
sequence encoding IEGR. In the PCR reaction, Pyrobest DNA
polymerase (manufactured by Takara Bio Inc.) was used, and the
reaction conditions were in accordance with the manufacturer's
recommended protocol. After restriction enzyme KpnI treatment, each
of these DNA fragments was inserted in a KpnI site of pPK4
described in JP-A Hei 9-322774 to thus obtain pPKK17Xa-PIns and
pPKK50Xa-PIns. The result of determining the base sequences of the
inserted fragments confirmed that the fusion genes were constructed
as expected. Note that the base sequences were determined using
BigDye.RTM. Terminator v3.1 Cycle Sequencing Kit (manufactured by
Applied BioSystems Inc.) and 3130 Genetic Analyzer (manufactured by
Applied BioSystems Inc.).
(v) Total Synthesis of Human Growth Hormone hGH Gene, and
Construction of Human Growth Hormone hGH-Secretory Expression
Plasmids in C. glutamicum
[0292] The amino acid sequence of human growth hormone (hGH) has
already been determined (Genbank Accession No. CAA23779.1). In
consideration of this sequence, particularly an amino acid sequence
of mature hGH excluding 26 residues of a signal sequence at the
N-terminus, and the codon usage in C. glutamicum, DNAs shown in
<SEQ ID NO: 56> to <SEQ ID NO: 69> were synthesized.
Using these DNAs as templates, and using independently synthesized
DNAs shown in <SEQ ID NO: 70> and <SEQ ID NO: 71> as
primers, a hGH gene was amplified by PCR. Thus, approximately 0.6
kbp of a DNA fragment shown in <SEQ ID NO: 72> was obtained.
The DNA fragment was inserted in a SmaI site of a cloning vector
pHSG398 (manufactured by Takara Bio Inc.) to thus obtain pHSG-hGH.
Using this pHSG-hGH as a template, and using the DNAs shown in
<SEQ ID NO: 70> and <SEQ ID NO: 71> as primers, a hGH
gene region was amplified by PCR. Thus, approximately 0.6 kbp of a
hGH gene fragment was obtained. Next, pPKSPTG1 described in
WO01/23591 (the pPKSPTG1 is a protransglutaminase (transglutaminase
having a pro-structural portion)-secretory expression vector
having: the promoter of the cspB gene derived from the C.
glutamicum ATCC13869 strain; a DNA encoding 25 amino acid residues
of a signal peptide of CspA (SlpA) <Genbank Accession No.
BAB62413.1> derived from a C. ammoniagenes ATCC6872 strain, the
DNA expressibly ligated downstream of the promoter; and the
protransglutaminase gene derived from S. mobaraense, the gene
ligated downstream of the DNA encoding the signal peptide in such a
manner as to be expressed in the form of a fusion protein with the
signal peptide) and pPKPTG1 described in WO01/23591 (containing the
promoter region of CspB derived from the C. glutamicum ATCC13869
strain and the DNA encoding the signal peptide) were used as
templates, and primers shown in <SEQ ID NO: 20> and <SEQ
ID NO: 73> or <SEQ ID NO: 20> and <SEQ ID NO: 74>
were used in PCR to amplify regions encoding the promoter region of
the C. glutamicum ATCC13869-derived CspB and the signal peptide of
the C. ammoniagenes ATCC6872 strain-derived CspA or the C.
glutamicum ATCC13869 strain-derived CspB. Thus, DNA fragments, each
approximately 0.7 kbp, were obtained. Then, using the two amplified
DNA fragments (the hGH gene fragment; the fragment of the region
encoding the CspB promoter region and any one of the signal
peptides) as templates, and using the DNAs shown in <SEQ ID NO:
20> and <SEQ ID NO: 71> as primers, DNA fragments, each
approximately 1.2 kbp, were obtained by PCR, in each of which the
two DNA fragments were fused together. Note that the primers of
<SEQ ID NO: 20> and <SEQ ID NO: 71> were each designed
to have the restriction enzyme KpnI recognition sequence, and the
primers of <SEQ ID NO: 73> and <SEQ ID NO: 74> were
each designed to have a sequence encoding amino acid residues at
the N-terminus of hGH so as to construct a fusion gene made of the
hGH gene and the region encoding the corresponding signal peptide.
In the PCR reaction, Pyrobest DNA polymerase (manufactured by
Takara Bio Inc.) was used, and the reaction conditions were in
accordance with the manufacturer's recommended protocol. After
restriction enzyme KpnI treatment, each of these DNA fragments was
inserted in a KpnI site of pPK4 described in JP-A Hei 9-322774 to
thus obtain pPS-hGH and pPK-hGH. The result of determining the base
sequences of the inserted fragments confirmed that the fusion genes
were constructed as expected. Note that all the base sequences were
determined using BigDye.RTM. Terminator v3.1 Cycle Sequencing Kit
(manufactured by Applied BioSystems Inc.) and 3130 Genetic Analyzer
(manufactured by Applied BioSystems Inc.).
(vi) Construction of Secretory Expression Plasmids for Human Growth
Hormone hGH Fused with Signal Peptide of Cell Surface Protein CspB
of C. glutamicum ATCC13869, Amino Acid Residues at N-Terminus of
the Mature Protein, and Factor Xa Protease Recognition Sequence
[0293] Using pPKK6Xa-PIns, pPKK17Xa-PIns, or pPKK50Xa-PIns
constructed above in (iv) as a template, and using corresponding
synthetic DNAs shown in <SEQ ID NO: 20> and <SEQ ID NO:
75>, <SEQ ID NO: 20> and <SEQ ID NO: 76>, or <SEQ
ID NO: 20> and <SEQ ID NO: 77> as primers, regions
encoding the CspB promoter region, the 30 amino acid residues of
the signal peptide at the N-terminus of CspB, the amino acid
residues (6, 17, or 50 residues) at the N-terminus of the CspB
mature protein, and the Factor Xa protease recognition sequence
(IEGR) were amplified by PCR. On the other hand, using the plasmid
pPS-hGH constructed above in (v) as a template, and using the
synthetic DNA shown in <SEQ ID NO: 70> and <SEQ ID NO:
71> as primers, a hGH gene region was amplified by PCR. Then,
using the two amplified DNA fragments (each fragment of the region
encoding the CspB promoter region, the CspB signal peptide, the
amino acid residues at the N-terminus of the mature CspB, and IEGR;
the hGH gene fragment) as templates, and using the DNAs shown in
<SEQ ID NO: 20> and <SEQ ID NO: 71> as primers, DNA
fragments were obtained by PCR, in each of which the two DNA
fragments were fused together. Note that the primers of <SEQ ID
NO: 20> and <SEQ ID NO: 71> were each designed to have the
restriction enzyme KpnI recognition sequence, and the primers of
<SEQ ID NO: 75>, <SEQ ID NO: 76>, and <SEQ ID NO:
77> were each designed to have a sequence encoding amino acid
residues at the N-terminus of hGH so as to construct a fusion gene
made of the hGH gene and the region encoding the Factor Xa protease
recognition sequence (IEGR). In the PCR reaction, Pyrobest DNA
polymerase (manufactured by Takara Bio Inc.) was used, and the
reaction conditions were in accordance with the manufacturer's
recommended protocol. After restriction enzyme KpnI treatment, each
of these DNA fragments was inserted in a KpnI site of pPK4
described in JP-A Hei 9-322774 to thus obtain pPKK6Xa-hGH,
pPKK17Xa-hGH, and pPKK50Xa-hGH.
(vii) Construction of Teriparatide-Secretory Expression Plasmid
pPKK6Xa-Teri
[0294] The amino acid sequence of a matured human parathyroid
hormone PTH has already been determined (Genbank Accession No.
AAA60215.1). A peptide from 1 to 34 residues at the N-terminus of
this human parathyroid hormone PTH is known as a peptide
teriparatide having a physiological activity serving as an
osteoporosis drug. In consideration of the amino acid sequence of
this teriparatide and the codon usage in C. glutamicum, DNAs shown
in <SEQ ID NO: 78> and <SEQ ID NO: 79> were
synthesized. Using the DNAs as templates, and using independently
synthesized DNAs shown in <SEQ ID NO: 80> and <SEQ ID NO:
81> as primers, a teriparatide gene shown in <SEQ ID NO:
82> was amplified by PCR. The DNA fragment was inserted in a
SmaI site of a cloning vector pHSG398 (manufactured by Takara Bio
Inc.) to thus obtain pHSG-Teri. Using this pHSG-Teri as a template,
and using the DNAs shown in <SEQ ID NO: 80> and <SEQ ID
NO: 81> as primers, a teriparatide gene region was amplified by
PCR. Next, pPKSPTG1 described in WO01/23591 (containing the
promoter region of CspB derived from the C. glutamicum ATCC13869
strain and the DNA encoding the signal peptide of CspA (SlpA)
derived from the C. ammoniagenes ATCC6872 strain) and pPKPTG1
described in WO01/23591 (containing the promoter region of CspB
derived from the C. glutamicum ATCC13869 strain and the DNA
encoding the signal peptide) were used as templates, and primers
shown in <SEQ ID NO: 20> and <SEQ ID NO: 83> or <SEQ
ID NO: 20> and <SEQ ID NO: 84> were used in PCR to amplify
regions encoding the promoter region of the C. glutamicum
ATCC13869-derived CspB and the signal peptide of the C.
ammoniagenes ATCC6872 strain-derived CspA or the C. glutamicum
ATCC13869 strain-derived CspB. Then, using the two amplified DNA
fragments (the teriparatide gene fragment; the fragment of the
region encoding the CspB promoter region and any one of the signal
peptides) as templates, and using the DNAs shown in <SEQ ID NO:
20> and <SEQ ID NO: 81> as primers, DNA fragments, each
approximately 0.8 kbp, were obtained by PCR, in each of which the
two DNA fragments were fused together. Note that the primers of
<SEQ ID NO: 20> and <SEQ ID NO: 81> were each designed
to have the restriction enzyme KpnI recognition sequence, and the
primers of <SEQ ID NO: 83> and <SEQ ID NO: 84> were
each designed to have a sequence encoding amino acid residues at
the N-terminus of teriparatide so as to construct a fusion gene
made of the teriparatide gene and the region encoding the
corresponding signal peptide. In the PCR reaction, Pyrobest DNA
polymerase (manufactured by Takara Bio Inc.) was used, and the
reaction conditions were in accordance with the manufacturer's
recommended protocol. After restriction enzyme KpnI treatment, each
of these DNA fragments was inserted in a KpnI site of pPK4
described in JP-A Hei 9-322774 to thus obtain pPS-Teri and
pPK-Teri.
[0295] Next, using pHSG-Teri described above as a template, and
using DNAs shown in <SEQ ID NO: 85> and <SEQ ID NO: 81>
as primers, a teriparatide gene region was amplified by PCR.
Moreover, using pPKK6Xa-hGH constructed above in (vi) as a
template, and using primers shown in <SEQ ID NO: 20> and
<SEQ ID NO: 86>, a region encoding the CspB promoter region,
the 30 amino acid residues of the signal peptide at the N-terminus
of CspB, the 6 amino acid residues at the N-terminus of the CspB
mature protein, and the Factor Xa protease recognition sequence
(IEGR) was amplified by PCR. Then, using the two amplified DNA
fragments (the teriparatide gene fragment; the fragment of the
region encoding the CspB promoter region, the CspB signal peptide,
the 6 amino acid residues at the N-terminus of the CspB mature
protein, and IEGR) as templates, and using the DNAs shown in
<SEQ ID NO: 20> and <SEQ ID NO: 81> as primers,
approximately 0.8 kbp of a DNA fragment was obtained by PCR, in
which the two DNA fragments were fused together. Note that the
primers of <SEQ ID NO: 20> and <SEQ ID NO: 81> were
each designed to have the restriction enzyme KpnI recognition
sequence, and the primer of <SEQ ID NO: 85> was designed to
have a sequence encoding the Factor Xa protease recognition
sequence (IEGR) so as to construct a fusion gene made of the
teriparatide gene and the region encoding the Factor Xa protease
recognition sequence (IEGR). In the PCR reaction, Pyrobest DNA
polymerase (manufactured by Takara Bio Inc.) was used, and the
reaction conditions were in accordance with the manufacturer's
recommended protocol. After restriction enzyme KpnI treatment, the
DNA fragment was inserted in a KpnI site of pPK4 described in JP-A
Hei 9-322774 to thus obtain pPKK6Xa-Teri. The result of determining
the base sequence of the inserted fragment confirmed that the
fusion gene was constructed as expected. Note that all the base
sequences were determined using BigDye.RTM. Terminator v3.1 Cycle
Sequencing Kit (manufactured by Applied BioSystems Inc.) and 3130
Genetic Analyzer (manufactured by Applied BioSystems Inc.).
(viii) Construction of Teriparatide-Secretory Expression Plasmid
pPKK50TEV-Teri
[0296] Using the plasmid pPKK50Xa-PIns constructed above in (iii)
as a template, and using synthetic DNAs shown in <SEQ ID NO:
20> and <SEQ ID NO: 87> as primers, a region encoding the
5'-upstream region containing the promoter region of CspB, the 30
amino acid residues of the signal peptide at the N-terminus, the 50
residues on the N-terminal side of the mature cell surface protein,
and the amino acid sequence ENLYFQ recognized by the ProTEV
protease was amplified by PCR. On the other hand, using the plasmid
pPKK6Xa-Teri constructed above in (vii) as a template, and using
synthetic DNAs shown in <SEQ ID NO: 88> and <SEQ ID NO:
89> as primers, a teriparatide gene region was amplified by PCR.
Then, using the amplified DNA fragments (the fragment of the region
encoding the CspB promoter, the CspB signal peptide, the 50
residues in the N-terminal amino acid sequence of CspB, and the
amino acid sequence ENLYFQ; the teriparatide gene fragment) as
templates, and using the DNAs shown in <SEQ ID NO: 20> and
<SEQ ID NO: 89> as primers, a DNA fragment was obtained by
PCR, in which the DNA fragments were fused together. Note that the
primers of <SEQ ID NO: 20> and <SEQ ID NO: 89> were
each designed to have the restriction enzyme
[0297] KpnI recognition sequence, and the primer of <SEQ ID NO:
88> was designed to have a sequence encoding an amino acid
sequence on the N-terminal side of teriparatide so as to construct
a fusion gene made of teriparatide and the base sequence encoding
ENLYFQ. In the PCR reaction, Pyrobest DNA polymerase (manufactured
by Takara Bio Inc.) was used, and the reaction conditions were in
accordance with the manufacturer's recommended protocol. After
restriction enzyme KpnI treatment, the DNA fragment was inserted in
a KpnI site of pPK4 described in JP-A Hei 9-322774 to thus obtain a
plasmid pPKK50TEV-Teri. The result of determining the base sequence
of the inserted fragment confirmed that the fusion gene was
constructed as expected. Note that the base sequence was determined
using BigDye.RTM. Terminator v3.1 Cycle Sequencing Kit
(manufactured by Applied BioSystems Inc.) and 3130 Genetic Analyzer
(manufactured by Applied BioSystems Inc.).
(2) Secretory Expression of Fusion Protein CspB50TEV-Teriparatide
(Abbreviated as 50-Teri) Using pPKK50TEV-Teri
[0298] Using pPKK50TEV-Teri constructed in (1), a C. glutamicum
YDK010 strain described in WO01/23591 was transformed. The obtained
transformed strain was cultured at 30.degree. C. for 72 hours in MM
liquid media (120 g of glucose, 0.4 g of magnesium sulfate
heptahydrate, 30 g of ammonium sulfate, 1 g of potassium dihydrogen
phosphate, 0.01 g of iron sulfate heptahydrate, 0.01 g of manganese
sulfate pentahydrate, 200 .mu.g of thiamine hydrochloride, 500
.mu.g of biotin, 0.15 g of DL-methionine, 50 g of calcium
carbonate, adjusted to 1 L with water and to pH 7.5) containing 25
mg/l of kanamycin. After the culturing was completed, each culture
solution was centrifuged. The resulting culture supernatant was
subjecting to reducing SDS-PAGE. Staining with CBB R-250
(manufactured by Bio-Rad Laboratories, Inc.) showed a band of the
target fusion protein 50-Teri.
[0299] On the other hand, the obtained transformed strain was
cultured, while being agitated for aeration, at 30.degree. C. for 3
days in a jar fermenter of 1 L capacity, in which 300 mL of a MMTG
liquid medium (120 g of glucose, 2 g of calcium chloride, 3 g of
magnesium sulfate heptahydrate, 3 g of ammonium sulfate, 1.5 g of
potassium dihydrogen phosphate, 0.03 g of iron sulfate
heptahydrate, 0.03 g of manganese sulfate pentahydrate, 450 dig of
thiamine hydrochloride, 450 .mu.g of biotin, 0.15 g of
DL-methionine, 50 g of calcium carbonate, adjusted to 1 L with
water and to pH 6.7) containing 25 mg/l of kanamycin had been
charged and the pH was being maintained at 6.7 by adding an ammonia
gas.
(3) Removal of Microbial Cells from Culture Solution, and Storage
of Microbial Cell-Removed Culture Solution
[0300] After the culturing was completed, the culture solution was
transferred to microtubes and centrifuged using a centrifuge at
12000 G for 10 minutes to separate the microbial cells. The
resulting centrifuged supernatant was filtered through a sterile
filter having a pore diameter of 0.22 .mu.m, and the resulting
filtrate was cryopreserved at -80.degree. C. as a "microbial
cell-removed culture solution" (corresponding to "solution obtained
in step (1)").
(4) Precipitation and Solubilization of Fusion Protein (50-Teri)
Due to pH Change
[0301] The cryopreserved microbial cell-removed culture solution
was thawed at 25.degree. C. and centrifuged using a centrifuge at
12000 G for 1 minute to separate a precipitate mainly including an
inorganic salt formed during the cryopreservation. To four
microtubes, 0, 5, 10, and 30 .mu.L of aqueous solutions of 0.5 M
sulfuric acid and 30, 25, 20, and 0 .mu.L of Milli Q water were
respectively added and uniformly mixed. Then, 100 .mu.L of the
resulting centrifuged supernatant was dispensed into each of the
tubes. All of the mixtures were adjusted to a volume of 130
.mu.L.
[0302] After stirring, these were left alone for 10 minutes.
Thereby, "pH-adjusted culture solutions" at pH 7.4, pH 6.6, pH 3.8,
and pH 1.7 were obtained (corresponding to "solution obtained in
step (2)"). The "pH-adjusted culture solutions" at pH 6.6, pH 3.8,
and pH 1.7 were clouded, and precipitation formation was observed.
Those "pH-adjusted culture solutions" were centrifuged using a
centrifuge at 12000 G for 5 minutes to separate precipitates formed
by the pH change (corresponding to "solid separated in step (3)").
The resulting centrifuged supernatants were transferred as
"supernatants of the pH-adjusted culture solutions" (corresponding
to "solution after solid separation in step (3)") to different
microtubes separately. To the remaining precipitates, a buffer (100
mM Tris-HCl, pH 8.5) was added in the same volume as those of the
centrifuged supernatants having been removed. By stirring, the
precipitates were immediately dissolved, and "precipitate-dissolved
solutions" were obtained (corresponding to "solution obtained in
step (4)"). All of the precipitate-dissolved solutions had a pH of
around neutral, which were pH 8.3, pH 8.3, pH 8.1, and pH 7.8. In
consideration of the above pH range of the precipitate formation
and the fact that the precipitates formed were reversibly and
immediately re-dissolved at around the neutral pH, it was found out
that the precipitation phenomenon observed in step (2) is totally
different from a phenomenon in which a protein is denatured by acid
generally in an irreversible manner.
[0303] The "supernatants of the pH-adjusted culture solutions" at
pH 7.4, pH 6.6, pH 3.8, and pH 1.7, and the "precipitate-dissolved
solutions" obtained by the above-described operations were
subjected to reducing SDS-PAGE, and bands of 50-Teri were checked
to analyze and evaluate the precipitation and solubilization of
50-Teri due to the pH change. Note that the reducing SDS-PAGE was
performed using Any kD.TM. Mini-PROTEAN.RTM. TGX.TM. Precast Gel
(Bio-Rad Laboratories, Inc.), and the bands of the fusion protein
50-Teri were detected by staining with SYPRO.RTM. Ruby (Life
Technologies Japan Ltd.). Subsequently, the band intensity at each
pH was quantified using software Multi Gauge (manufactured by
FUJIFILM Corporation), and the recovery ratio of 50-Teri at each pH
was calculated according to the following equation:
the recovery ratio (%)=[a band intensity of the fusion protein in
the solution obtained in step (4)/{the band intensity of the fusion
protein in the solution obtained in step (4)+a band intensity of
the fusion protein in the solution after solid separation in step
(3)}].times.100.
[0304] It should be noted that, in Examples and Comparative Example
to be described below also, the recovery ratio in reducing SDS-PAGE
was calculated in the same manner as in Example 1.
[0305] The calculated recovery ratios of 50-Teri from the
"pH-adjusted culture solutions" at pH 7.4, pH 6.6, pH 3.8, and pH
1.7 were respectively 1%, 95%, 99%, and 99%. FIG. 1-A shows the
relation between the calculated recovery ratio and the pH of the
"pH-adjusted culture solutions." The graph revealed that the
precipitation phenomenon observed in step (2) is totally different
from an isoelectric point precipitation phenomenon in which the
solubility of a protein becomes lowest at an isoelectric point
thereof.
[0306] FIG. 1-B shows the pH of the "pH-adjusted culture
solutions," band portions of the fusion protein 50-Teri in images
of electrophoresis of the "supernatants of the pH-adjusted culture
solutions" and the corresponding "precipitate-dissolved solutions,"
and the recovery ratio.
[0307] The 50-Teri was present in the "supernatant of the
pH-adjusted culture solution" at pH 7.4. The 50-Teri was not
detected in the "supernatants of the pH-adjusted culture solutions"
at pH 6.6, pH 3.8, and pH 1.7, but was detected in the
corresponding "precipitate-dissolved solutions" at these pHs.
Example 1-2
Production of Fusion Protein 50-Teri Having Bioactive Peptide
Teriparatide, and Production of Target Protein Teriparatide
[0308] In Example 1-2, in the same manner as in Example 1, a
bioactive peptide teriparatide (34 amino acid residues) (Teri) was
used as a target protein, a sequence consisting of 50 amino acid
residues from the N-terminus of a CspB mature protein (CspB50) was
used as a protein having a self-assembly capability, a proTEV
protease recognition sequence was used as an amino acid sequence
used for an enzymatic cleavage, and a coryneform bacterium C.
glutamicum was used as a host.
(1) Construction of Teriparatide-Secretory Expression Plasmid
pPKK50TEV-Teri
[0309] constructed according to the procedure described in Example
1 (1).
(2) Secretory Expression of Fusion Protein Using pPKK50TEV-Teri
[0310] According to the procedure described in Example 1 (2), a C.
glutamicum YDK010 strain transformed using pPKK50TEV-Teri was
cultured.
(3) Removal of Microbial Cells from Culture Solution, and Storage
of Microbial Cell-Removed Culture Solution
[0311] carried out according to the procedure described in Example
1 (3).
(4) Precipitation and Solubilization of Fusion Protein 50-Teri due
to pH Change
[0312] The cryopreserved microbial cell-removed culture solution
was thawed at 25.degree. C. and centrifuged using a centrifuge at
12000 G for 1 minute to separate a precipitate mainly including an
inorganic salt formed during the cryopreservation. To eight
microtubes, 0 .mu.L, 5 .mu.L, 10 .mu.L, 15 .mu.L, 20 .mu.L, 30
.mu.L, 40 .mu.L, and 50 .mu.L of aqueous solutions of 0.5 M
sulfuric acid and 50 .mu.L, 45 .mu.L, 40 .mu.L, 25 .mu.L, 30 .mu.L,
20 .mu.L, 10 .mu.L, and 0 .mu.L of Milli Q water were respectively
added and uniformly mixed. Then, 600 .mu.L of the resulting
centrifuged supernatant was dispensed into each of the tubes. All
of the mixtures were adjusted to a volume of 650 .mu.L.
[0313] After stirring, these were left alone for 10 minutes.
Thereby, "pH-adjusted culture solutions" at pH 7.9, pH 7.6, pH 7.3,
pH 7.1, pH 6.6, pH 4.9, pH 4.1, and pH 3.7 were obtained
(corresponding to "solution obtained in step (2)"). The
"pH-adjusted culture solutions" at pH 7.1, pH 6.6, pH 4.9, pH 4.1,
and pH 3.7 were clouded, and precipitation formation was observed.
Those "pH-adjusted culture solutions" were centrifuged using a
centrifuge at 12000 G for 5 minutes to separate precipitates formed
by the pH change (corresponding to "solid separated in step (3)").
The resulting centrifuged supernatants were transferred as
"supernatants of the pH-adjusted culture solutions" (corresponding
to "solution after solid separation in step (3)") to different
microtubes separately. To the remaining precipitates, a buffer (100
mM Tris-HCl, pH 8.5) was added in the same volume as those of the
centrifuged supernatants having been removed. By stirring, the
precipitates were immediately dissolved, and "precipitate-dissolved
solutions" were obtained (corresponding to "solution obtained in
step (4)"). All of the "precipitate-dissolved solutions" had a pH
of around neutral, which were pH 8.0, pH 8.0, pH 8.0, pH 8.0, pH
8.0, pH 7.9, pH 7.7, and pH 7.6.
[0314] The "supernatants of the pH-adjusted culture solutions" at
pH 7.9, pH 7.6, pH 7.3, pH 7.1, pH 6.6, pH 4.9, pH 4.1, and pH 3.7,
and the "precipitate-dissolved solutions" obtained by the
above-described operations were subjected to reducing SDS-PAGE and
reversed-phase HPLC to analyze and evaluate the precipitation and
solubilization of 50-Teri due to the pH change. Note that the
recovery ratio of 50-Teri was calculated according to the following
equation using reversed-phase HPLC:
the recovery ratio (%)=[an amount of the fusion protein in the
solution obtained in step (4)/{the amount of the fusion protein in
the solution obtained in step (4)+an amount of the fusion protein
in the solution after solid separation in step (3)}].times.100.
[0315] The amounts of the fusion proteins were quantified from the
peak area in the reversed-phase HPLC. Specifically, a calibration
curve was created using a known substance (IGF-1), and the peak
area of each measurement sample was assigned to the calibration
curve, so that the amount of the fusion protein was calculated. The
conditions of the reversed-phase HPLC are shown below.
System: a set of Waters Alliance PDA system Column: YMC-Triart C18
.phi.4.6.times.100 mm, a particle diameter of 5 .mu.m, a pore
diameter of 12 nm Column temp.: 30.degree. C. Mobile phase A:
aqueous solution of 10 mM ammonium acetate, aqueous solution of 10%
acetonitrile, pH 7.0 Mobile phase B: aqueous solution of 10 mM
ammonium acetate, aqueous solution of 80% acetonitrile, pH 7.0 Flow
rate: 1.0 mL/min
Detection: 220 nm
[0316] Injection volume: 30 .mu.L
TABLE-US-00002 Gradient: Time (min) A (%) B (%) 0 100 0 25 50 50 28
0 100
[0317] The recovery ratios of 50-Teri from the "pH-adjusted culture
solutions" at pH 7.9, pH 7.6, pH 7.3, pH 7.1, pH 6.6, pH 4.9, pH
4.1, and pH 3.7 were respectively 0%, 0%, 0%, 63%, 95%, 96%, 98%,
and 100%. FIG. 1-2A shows a relation graph between the calculated
recovery ratio and the pH of the "pH-adjusted culture
solutions."
[0318] FIG. 1-2B shows images of electrophoresis of the
"pH-adjusted culture solutions" and the corresponding
"precipitate-dissolved solutions," and photographs of extracted
band portions of the fusion protein 50-Teri.
[0319] The 50-Teri was present in the "supernatants of the
pH-adjusted culture solutions" at pH 7.9, pH 7.6, pH 7.3, and pH
7.1. Although the 50-Teri was not detected in the "supernatants of
the pH-adjusted culture solutions" at pH 6.6, pH 4.9, pH 4.1, and
pH 3.7, significant amounts thereof were detected in the
corresponding "precipitate-dissolved solutions" at these pHs.
[0320] Moreover, the "supernatant of the pH-adjusted culture
solution" at pH 4.9 and the corresponding "precipitate-dissolved
solution" were subjected to a reversed-phase HPLC analysis to
compare the fusion protein 50-Teri, impurities, and the like
contained in the two. As a result, a large amount of impurities
(for example, a peak group having a retention time of 0 minutes to
5 minutes) derived from the culture solution were detected in the
"supernatant of the pH-adjusted culture solution"; meanwhile,
impurity peaks other than the target fusion protein 50-Teri were
hardly detected in the "precipitate-dissolved solution" (FIG.
1-2C). This revealed that the liquid-solid separation of the fusion
protein by precipitation according to the present invention is
applicable as a partial purification process.
(5) Enzymatic Cleavage of Fusion Protein 50-Teri, and Acquisition
of Target Protein Teriparatide
[0321] The precipitated fusion protein 50-Teri obtained from the
"pH-adjusted culture solution" having a pH adjusted to 4.9 in (4)
above was washed with a 20 mM Tris HCl buffer (pH 5.0) to remove
the culture solution attached to the precipitate. Next, the
precipitate was dissolved into a buffer of 6 M urea+20 mM Tris HCl
(pH 8.0), and a "precipitate-dissolved solution" was prepared. The
"precipitate-dissolved solution" was diluted 10-fold by adding
ultrapure water thereto, and then a ProTEV protease (capable of
recognizing the amino acid sequence ENLYFQ in the fusion protein,
Promega Corporation, V6102) was added to the resultant. Thereby,
the fusion protein was enzymatically cleaved into CspB50TEV
containing the protein having a self-assembly capability and the
target protein teriparatide. Thus, an "enzymatic cleavage solution"
was obtained. As a result of analyzing the enzymatic cleavage
solution by reversed-phase HPLC, a peak believed to be of
teriparatide was detected (FIG. 1-2D).
[0322] Subsequently, in order to confirm that the substance formed
in the enzymatic cleavage solution was teriparatide, the "enzymatic
cleavage solution" was subjected to reversed-phase HPLC. An eluate
was obtained at a retention time of around 18.6 minutes under the
above-described conditions of the reversed-phase HPLC, and then the
substance believed to be teriparatide was purified. The purified
substance was subjected to an N-terminal amino acid sequence
analysis and mass spectrometry, and compared with standard
Teriparatide (BACHEM, cat #H-4835).
[0323] The N-terminal amino acid sequence analysis was performed
using a protein sequencer PPSQ-10 (manufactured by Shimadzu
Corporation) based on the Edman degradation in accordance with the
method (instruction manual) recommended by Shimadzu Corporation.
The mass spectrometry was performed using AXIMA-TOF2 (manufactured
by Shimadzu Corporation) based on MALDI-TOF-MS in accordance with
the method (instruction manual) recommended by Shimadzu
Corporation.
[0324] As a result of the N-terminal amino acid sequence analysis,
10 amino acid residues on the N-terminal side of the purified
substance matched 10 amino acid residues on the N-terminal side of
standard Teriparatide.
[0325] As a result of the mass spectrometry, when the purified
substance was measured, the measurement mass of 4118.9 (the
measurement error was .+-.0.1%) was detected; when standard
Teriparatide was measured, the measurement mass of 4118.1 (the
measurement error was .+-.0.1%) was detected (FIG. 1-2E). In other
words, the measurement mass of the purified substance matched the
measurement mass of standard Teriparatide. It was revealed that the
purified substance was the target protein teriparatide. The purity
of teriparatide obtained in this Example was 94%, which was
calculated based on a peak area detected using reversed-phase HPLC
according to the following equation:
the purity (%)=(the peak area of teriparatide/a total of all peak
areas).times.100.
[0326] Incidentally, the reversed-phase HPLC was carried out under
the following conditions.
System: a set of Waters Alliance PDA system Column: YMC-Pack C8
.phi.4.6.times.100 mm, particle diameter 5 .mu.m, a pore diameter
of 30 nm Column temp.: 30.degree. C. Mobile phase A: aqueous
solution of 10 mM ammonium acetate, aqueous solution of 10%
acetonitrile, pH 7.0 Mobile phase B: aqueous solution of 10 mM
ammonium acetate, aqueous solution of 80% acetonitrile, pH 7.0 Flow
rate: 1.0 mL/min
Detection: 220 nm
[0327] Injection volume: 30 .mu.L
TABLE-US-00003 Gradient: Time (min) A (%) B (%) 0 100 0 60 0
100
[0328] These results confirmed that the purified substance was
teriparatide, and that the target protein can be obtained from the
fusion protein.
Example 2
Production of Fusion Protein (CspB50Lys-Bivalirudin18 (Abbreviated
as 50-Biva18)) Having Portion of Bioactive Peptide Bivalirudin, and
Production of Target Protein Bivalirudin18 (Abbreviated as
Biva18)
[0329] In Example 2, a portion (18 amino acid residues) (Biva18) of
a bioactive peptide bivalirudin was used as a target protein, a
sequence consisting of 50 amino acid residues from the N-terminus
of a CspB mature protein (CspB50) was used as a protein having a
self-assembly capability, and a coryneform bacterium C. glutamicum
was used as a host.
(1) Construction of Biva18-Secretory Expression Plasmid
pPKK50Lys-Biva18 in C. glutamicum
[0330] A bioactive peptide bivalirudin known as an anticoagulant
having a thrombin inhibitory activity is a peptide consisting of 20
residues in full length and having a D-phenylalanine residue at the
N-terminus. In consideration of an amino acid sequence of
18-residue peptides (=Biva18) excluding an L-proline residue and
the D-phenylalanine residue at the N-terminus and the codon usage
in C. glutamicum, a total synthesis of <SEQ ID NO: 90>
containing the Biva18 gene was performed.
[0331] Next, using the plasmid pPKK50Xa-PIns described in Example 1
(1) (iii) as a template, and using synthetic DNAs shown in <SEQ
ID NO: 20> and <SEQ ID NO: 91> as primers, a region
encoding the 5'-upstream region containing the promoter region of
CspB, the 30 amino acid residues of the signal peptide at the
N-terminus, the 50 residues on the N-terminal side of the mature
cell surface protein, and a lysine residue was amplified by PCR.
Then, using the amplified DNA fragment (the fragment of the region
encoding the CspB promoter, the CspB signal peptide, the 50
residues in the N-terminal amino acid sequence of CspB, and the
lysine residue) and the Biva18 gene fragment <SEQ ID NO: 90>
as templates, and using DNAs shown in <SEQ ID NO: 20> and
<SEQ ID NO: 92> as primers, a DNA fragment was obtained by
PCR, in which the two DNA fragments were fused together. Note that
the primers of <SEQ ID NO: 20> and <SEQ ID NO: 92> were
each designed to have the restriction enzyme KpnI recognition
sequence, and the primer of <SEQ ID NO: 91> was designed to
have a sequence encoding the amino acid sequence on the N-terminal
side of Biva18 so as to construct a fusion gene made of Biva18 and
the base sequence encoding the 50 residues in the N-terminal amino
acid sequence of CspB and the lysine residue. In the PCR reaction,
Pyrobest DNA polymerase (manufactured by Takara Bio Inc.) was used,
and the reaction conditions were in accordance with the
manufacturer's recommended protocol. After restriction enzyme KpnI
treatment, the DNA fragment was inserted in a KpnI site of pPK4
described in JP-A Hei 9-322774 to thus obtain a plasmid
pPKK50Lys-Biva18 The result of determining the base sequence of the
inserted fragment confirmed that the fusion gene was constructed as
expected. Note that the base sequence was determined using
BigDye.RTM. Terminator v3.1 Cycle Sequencing Kit (manufactured by
Applied BioSystems Inc.) and 3130 Genetic Analyzer (manufactured by
Applied BioSystems Inc.).
(2) Secretory Expression of Fusion Protein CspB50Lys-Bivalirudin18
(Abbreviated as 50-Biva18) Using pPKK50Lys-Biva18
[0332] Using pPKK50Lys-Biva18 constructed in (1), a C. glutamicum
YDK010 strain described in WO01/23591 was transformed. The obtained
transformed strain was cultured at 30.degree. C. for 72 hours in MM
liquid media (120 g of glucose, 0.4 g of magnesium sulfate
heptahydrate, 30 g of ammonium sulfate, 1 g of potassium dihydrogen
phosphate, 0.01 g of iron sulfate heptahydrate, 0.01 g of manganese
sulfate pentahydrate, 200 .mu.g of thiamine hydrochloride, 500
.mu.g of biotin, 0.15 g of DL-methionine, 50 g of calcium
carbonate, adjusted to 1 L with water and to pH 7.5) containing 25
mg/l of kanamycin. After the culturing was completed, each culture
solution was centrifuged. The resulting culture supernatant was
subjected to reducing SDS-PAGE. Staining with CBB R-250
(manufactured by Bio-Rad Laboratories, Inc.) showed a band of the
target protein Bivalirudin18.
[0333] On the other hand, the obtained transformed strain was
cultured, while being agitated for aeration, at 30.degree. C. for 3
days in a jar fermenter of 1 L capacity, in which 300 mL of a MMTG
liquid medium (120 g of glucose, 2 g of calcium chloride, 3 g of
magnesium sulfate heptahydrate, 3 g of ammonium sulfate, 1.5 g of
potassium dihydrogen phosphate, 0.03 g of iron sulfate
heptahydrate, 0.03 g of manganese sulfate pentahydrate, 450 .mu.g
of thiamine hydrochloride, 450 .mu.g of biotin, 0.15 g of
DL-methionine, 50 g of calcium carbonate, adjusted to 1 L with
water and to pH 6.7) containing 25 mg/l of kanamycin had been
charged and the pH was being maintained at 6.7 by adding an ammonia
gas. After the culturing was completed, each culture solution was
centrifuged. The resulting culture supernatant was subjected to
reducing SDS-PAGE. Staining with CBB R-250 (manufactured by Bio-Rad
Laboratories, Inc.) showed a band of the target protein.
(3) Removal of Microbial Cells from Culture Solution, and Storage
of Microbial Cell-Removed Culture Solution
[0334] carried out in the same manner as in Example 1.
(4) Precipitation and Solubilization of Fusion Protein 50-Biva18
due to pH Change
[0335] The cryopreserved microbial cell-removed culture solution
was thawed at 25.degree. C. and centrifuged using a centrifuge at
12000 G for 1 minute to separate a precipitate mainly including an
inorganic salt formed during the cryopreservation. To four
microtubes, 0, 5, 10, and 30 .mu.L of aqueous solutions of 0.5 M
sulfuric acid and 30, 25, 20, and 0 .mu.L of Milli Q water were
respectively added and uniformly mixed. Then, 100 .mu.L of the
resulting centrifuged supernatant was dispensed into each of the
tubes. All of the mixtures were adjusted to a volume of 130
.mu.L.
[0336] After stirring, these were left alone for 10 minutes.
Thereby, "pH-adjusted culture solutions" at pH 7.8, pH 4.7, pH 2.9,
and pH 1.6 were obtained (corresponding to "solution obtained in
step (2)"). The "pH-adjusted culture solutions" at pH 2.9 and pH
1.6 were clouded, and precipitation formation was observed. Those
"pH-adjusted culture solutions" were centrifuged using a centrifuge
at 12000 G for 5 minutes to separate precipitates formed by the pH
change (corresponding to "solid separated in step (3)"). The
resulting centrifuged supernatants were transferred as
"supernatants of the pH-adjusted culture solutions" (corresponding
to "solution after solid separation in step (3)") to different
microtubes separately. To the separated precipitates, a buffer (100
mM Tris-HCl, pH 8.5) was added in the same volume as those of the
supernatants of the pH-adjusted culture solutions. By stirring, the
precipitates were immediately dissolved, and "precipitate-dissolved
solutions" were obtained (corresponding to "solution obtained in
step (4)"). All of the "precipitate-dissolved solutions" had a pH
of around neutral, which were pH 8.3, pH 8.3, pH 8.1, and pH
7.8.
[0337] The "supernatants of the pH-adjusted culture solutions" at
pH 7.8, pH 4.7, pH 2.9, and pH 1.6, and the "precipitate-dissolved
solutions" obtained by the above-described operations were
subjected to reducing SDS-PAGE, and bands of the fusion protein
50-Biva18 were checked to evaluate the precipitation and
solubilization of the fusion protein 50-Biva18 due to the pH
change.
[0338] The calculated recovery ratios of 50-Biva18 from the
"pH-adjusted culture solutions" at pH 7.8, pH 4.7, pH 2.9, and pH
1.6 were respectively 3%, 5%, 65%, and 63%. FIG. 2-A shows the
relation between the calculated recovery ratio and the pH of the
"pH-adjusted culture solutions."
[0339] FIG. 2-B shows the pH of the "pH-adjusted culture
solutions," band portions of the fusion protein 50-Biva18 in images
of electrophoresis of the "supernatants of the pH-adjusted culture
solutions" and the corresponding "precipitate-dissolved solutions,"
and the recovery ratio.
[0340] The 50-Biva18 was present in the "supernatants of the
pH-adjusted culture solutions" at pH 7.8 and pH 4.7. Weak bands
thereof were detected in the "supernatants of the pH-adjusted
culture solutions" at pH 2.9 and pH 1.6, while strong bands were
detected in the "precipitate-dissolved solutions" at these pHs.
[0341] Next, by the same operations as above, a "pH-adjusted
culture solution" having a pH of 3.0 lower than the pH value of
approximately 4.5 at which the recovery ratio of 10% is achieved
according to FIG. 2-A (the pH is the upper limit of the pH
applicable in step (2)) was prepared and centrifuged. The resulting
"supernatant of the pH-adjusted culture solution" and a
corresponding "precipitate-dissolved solution" were subjected to a
reversed-phase HPLC analysis to compare the fusion protein
50-Biva18, impurities, and the like contained in the two. As a
result, a large amount of impurities (for example, a peak group
having a retention time of 0 minutes to 10 minutes) derived from
the culture solution were detected in the "supernatant of the
pH-adjusted culture solution"; meanwhile, impurity peaks other than
the target fusion protein 50-Biva18 were hardly detected in the
"precipitate-dissolved solution" (FIG. 2-C).
[0342] Incidentally, the reversed-phase HPLC was carried out under
the following conditions.
System: a set of Waters Alliance PDA system Column: YMC-Triart C18
.phi.4.6.times.100 mm, a particle diameter of 5 .mu.m, a pore
diameter of 12 nm Column temp.: 30.degree. C. Mobile phase A:
aqueous solution of 10 mM ammonium acetate, aqueous solution of 10%
acetonitrile, pH 7.0 Mobile phase B: aqueous solution of 10 mM
ammonium acetate, aqueous solution of 80% acetonitrile, pH 7.0 Flow
rate: 1.0 mL/min
Detection: 220 nm
[0343] Injection volume: 30 .mu.L
TABLE-US-00004 Gradient: Time (min) A (%) B (%) 0 100 0 25 50 50 28
0 100
(5) Enzymatic Cleavage of Fusion Protein 50-Biva18, and Acquisition
of Target Protein Biva18
[0344] The precipitated fusion protein 50-Biva18 obtained from the
"pH-adjusted culture solution" having a pH adjusted to 3.0 in (4)
above was washed with a sulfuric acid solution of pH 3.0 to remove
the culture solution attached to the precipitate. Next, the
precipitate was dissolved into a buffer of 50 mM sodium bicarbonate
(pH 8.3), and a "precipitate-dissolved solution" was prepared. To
the precipitate-dissolved solution, trypsin (capable of recognizing
the amino acid sequence Lys in the fusion protein, SIGMA-ALDRICH
Co., T-303-10G) was added. Thus, an "enzymatic cleavage solution"
was obtained. As a result of subjecting the enzymatic cleavage
solution to reversed-phase HPLC, the peak of the fusion protein
50-Biva18, which was observed before the enzyme addition, was not
detected; instead, other peaks were newly detected. This revealed
that the cleavage reaction had favorably progressed (FIG. 2-D).
[0345] Subsequently, in order to confirm that the substance formed
in the enzymatic cleavage solution was Biva18, the "enzymatic
cleavage solution" was subjected to reversed-phase HPLC. An eluate
was obtained at a retention time of around 5 minutes, and then the
substance believed to be Biva18 was purified.
[0346] As a result of subjecting this purified substance to mass
spectrometry to measure the mass, the measurement mass of 1935.8 at
monoisotopic m/z (the measurement error was .+-.0.1%) was detected.
On the other hand, the theoretical monoisotopic m/z calculated from
the amino acid sequence of Biva18 was 1935.9, which was obtained as
a result of inputting the sequence of Biva18 to MS-Isotope
(http://prospector.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msisotope)-
. It was confirmed that this matched the measurement mass of 1935.8
detected from the purified substance (the measurement error was
.+-.0.1%) (FIG. 2-E). In other words, it was confirmed that the
purified substance was the target protein Biva18.
[0347] The purity of Biva18 obtained in this Example was 92%, which
was calculated based on a peak area detected using reversed-phase
HPLC according to the following equation:
the purity (%)=(the peak area of Biva18/a total of all peak
areas).times.100
[0348] Incidentally, the reversed-phase HPLC was carried out under
the following conditions.
System: a set of Waters Alliance PDA system Column: YMC-Triart C18
.phi.4.6.times.100 mm, a particle diameter of 5 .mu.m, a pore
diameter of 12 nm Column temp.: 30.degree. C. Mobile phase A:
aqueous solution of 10 mM ammonium acetate, aqueous solution of 10%
acetonitrile, pH 7.0 Mobile phase B: aqueous solution of 10 mM
ammonium acetate, aqueous solution of 80% acetonitrile, pH 7.0 Flow
rate: 1.0 mL/min
Detection: 220 nm
[0349] Injection volume: 30 .mu.L
TABLE-US-00005 Gradient: Time (min) A (%) B (%) 0 100 0 25 50 50 28
0 100
[0350] These results confirmed that the purified substance was
Biva18, and that the target protein can be obtained from the fusion
protein.
[0351] Next, the "enzymatic cleavage solution" was subjected to
strong anion exchange resin chromatography in place of the
reversed-phase HPLC, and the Biva18 was purified.
<Chromatography Conditions>
[0352] Column: strong anion exchange resin (HiTrap Q FF, 1 mL, GE
healthcare) A buffer (binding): aqueous solution of 25 mM Na
phosphate, pH 7.0 B buffer (elution): aqueous solution of 250 mM Na
phosphate, pH 7.0 Flow rate: 1 mL/min
Detection: 280 nm
[0353] Amount of sample having been loaded: 0.3 mg-Biva18 (prepared
with 250 .mu.L of the enzymatic cleavage solution and 750 .mu.L of
A buffer) Gradient elution: linear gradient, 0-100% B over 20
Column
Volumes (CV)
[0354] As a result of the strong anion chromatography, all Biva18
contained in the enzymatic cleavage solution adsorbed to the resin.
In the subsequent gradient elution, an eluate was obtained around
95% B and Biva18 was purified (FIG. 2-F). The purity of Biva18 thus
obtained was 83%, which was calculated based on a peak area
detected using reversed-phase HPLC according to the aforementioned
equation (FIG. 2-G).
Example 3
Production of Fusion Protein CspB50TEV-Proinsulin (abbreviated as
50-PIns) Having Proinsulin
[0355] In Example 3, proinsulin (86 amino acid residues) (PIns),
which is a proprotein of a bioactive peptide insulin, was used as a
target protein, a sequence consisting of 50 amino acid residues
from the N-terminus of a CspB mature protein (CspB50) was used as a
protein having a self-assembly capability, and a coryneform
bacterium C. glutamicum was used as a host.
(1) Secretory Expression of Fusion Protein 50-PIns Using
pPKK50PIns
[0356] Using the plasmid pPKK50PIns described in Example 1 (1)
(ii), a C. glutamicum YDK010 strain described in WO01/23591 was
transformed. The obtained transformed strain was cultured at
30.degree. C. for 72 hours in MM liquid media (120 g of glucose,
0.4 g of magnesium sulfate heptahydrate, 30 g of ammonium sulfate,
1 g of potassium dihydrogen phosphate, 0.01 g of iron sulfate
heptahydrate, 0.01 g of manganese sulfate pentahydrate, 200 .mu.g
of thiamine hydrochloride, 500 .mu.g of biotin, 0.15 g of
DL-methionine, 50 g of calcium carbonate, adjusted to 1 L with
water and to pH 7.5) containing 25 mg/l of kanamycin. After the
culturing was completed, each culture solution was centrifuged. The
resulting culture supernatant was subjected to reducing SDS-PAGE.
Staining with CBB R-250 (manufactured by Bio-Rad Laboratories,
Inc.) showed a band of the target protein.
(2) Removal of Microbial Cells from Culture Solution, and Storage
of Microbial Cell-Removed Culture Solution
[0357] carried out in the same manner as in Example 1.
(3) Precipitation and Solubilization of Fusion Protein 50-PIns due
to pH Change
[0358] The cryopreserved microbial cell-removed culture solution
was thawed at 25.degree. C. and centrifuged using a centrifuge at
12000 G for 1 minute to separate a precipitate formed during the
cryopreservation. To four microtubes, 0, 5, 10, and 30 .mu.L of
aqueous solutions of 0.5 M sulfuric acid and 30, 25, 20, and 0
.mu.L of Milli Q water were respectively added and uniformly mixed.
Then, 100 .mu.L of the resulting centrifuged supernatant was
dispensed into each of the tubes. All of the mixtures were adjusted
to a volume of 130 .mu.L.
[0359] After stirring, these were left alone for 10 minutes.
Thereby, "pH-adjusted culture solutions" at pH 7.8, pH 4.8, pH 4.0,
and pH 2.0 were obtained (corresponding to "solution obtained in
step (2)"). The "pH-adjusted culture solutions" at pH 4.8, pH 4.0,
and pH 2.0 were clouded, and precipitation formation was observed.
Those "pH-adjusted culture solutions" were centrifuged using a
centrifuge at 12000 G for 5 minutes to separate precipitates formed
by the pH change (corresponding to "solid separated in step (3)").
The resulting centrifuged supernatants were transferred as
"supernatants of the pH-adjusted culture solutions" (corresponding
to "solution after solid separation in step (3)") to different
microtubes separately. To the remaining precipitates, a buffer (100
mM Tris-HCl, pH 8.5) was added in the same volume as those of the
centrifuged supernatants having been removed. By stirring, the
precipitates were immediately dissolved, and "precipitate-dissolved
solutions" were obtained (corresponding to "solution obtained in
step (4)"); however, the precipitate formed at pH 2.0 was partially
insoluble. All of the "precipitate-dissolved solutions" had a pH of
around neutral, which were pH 8.3, pH 8.3, pH 8.3, and pH 8.3.
[0360] The "supernatants of the pH-adjusted culture solutions" at
pH 7.8, pH 4.8, pH 4.0, and pH 2.0, and the "precipitate-dissolved
solutions" obtained by the above-described operations were
subjected to reducing SDS-PAGE, and bands of 50-PIns were checked
to evaluate the precipitation and solubilization of 50-PIns due to
the pH change.
[0361] The calculated recovery ratios of 50-PIns from the
"pH-adjusted culture solutions" at pH 7.8, pH 4.8, pH 4.0, and pH
2.0 were respectively 1%, 54%, 58%, and 60%. FIG. 3-A shows the
relation between the calculated recovery ratio and the pH of the
"pH-adjusted culture solutions."
[0362] FIG. 3-B shows the pH of the "pH-adjusted culture
solutions," band portions of the fusion protein 50-PIns in images
of electrophoresis of the "supernatants of the pH-adjusted culture
solutions" and the corresponding "precipitate-dissolved solutions,"
and the recovery ratio.
[0363] The 50-PIns was present in the "supernatant of the
pH-adjusted culture solution" at pH 7.8. Weak bands thereof were
detected in the "supernatants of the pH-adjusted culture solutions"
at pH 4.8, pH 4.0, and pH 2.0, while strong bands were detected in
the "precipitate-dissolved solutions" at these pHs.
Comparative Example 1
Production of Proinsulin without Using Protein Having Self-Assembly
Capability
[0364] In Comparative Example 1, proinsulin (86 amino acid
residues) (PIns) was used as a target protein, and a coryneform
bacterium C. glutamicum was used as a host. Nevertheless, no
protein having a self-assembly capability was used.
(1) Secretory Expression of Proinsulin Using pPK-PIns
[0365] Using the plasmid pPKPIns described in Example 1 (1) (i), a
C. glutamicum YDK010 strain described in WO01/23591 was
transformed. The obtained transformed strain was cultured at
30.degree. C. for 72 hours in MM liquid media (120 g of glucose,
0.4 g of magnesium sulfate heptahydrate, 30 g of ammonium sulfate,
1 g of potassium dihydrogen phosphate, 0.01 g of iron sulfate
heptahydrate, 0.01 g of manganese sulfate pentahydrate, 200 .mu.g
of thiamine hydrochloride, 500 .mu.g of biotin, 0.15 g of
DL-methionine, 50 g of calcium carbonate, adjusted to 1 L with
water and to pH 7.5) containing 25 mg/l of kanamycin. After the
culturing was completed, each culture solution was centrifuged. The
resulting culture supernatant was subjected to reducing SDS-PAGE.
Staining with CBB R-250 (manufactured by Bio-Rad Laboratories,
Inc.) showed a band of the target protein.
(2) Removal of Microbial Cells from Culture Solution, and Storage
of Microbial Cell-Removed Culture Solution
[0366] carried out in the same manner as in Example 1.
(3) Precipitation and Solubilization of Proinsulin (PIns) due to pH
Change
[0367] The cryopreserved microbial cell-removed culture solution
was thawed at 25.degree. C. and centrifuged using a centrifuge at
12000 G for 1 minute to separate a precipitate formed during the
cryopreservation. To four microtubes, 0, 5, 10, and 30 .mu.L of
aqueous solutions of 0.5 M sulfuric acid and 30, 25, 20, and 0
.mu.L of Milli Q water were respectively added and uniformly mixed.
Then, 100 .mu.L of the resulting centrifuged supernatant was
dispensed into each of the tubes. All of the mixtures were adjusted
to a volume of 130 .mu.L.
[0368] After stirring, these were left alone for 10 minutes.
Thereby, "pH-adjusted culture solutions" at pH 7.5, pH 4.6, pH 4.0,
and pH 2.2 were obtained. The "pH-adjusted culture solution" at pH
2.2 was clouded, and precipitation formation was observed. Those
"pH-adjusted culture solutions" were centrifuged using a centrifuge
at 12000 G for 5 minutes to separate precipitates formed by the pH
change. The resulting centrifuged supernatants were transferred as
"supernatants of the pH-adjusted culture solutions" to different
microtubes separately. To the remaining precipitates, a buffer (100
mM Tris-HCl, pH 8.5) was added in the same volume as those of the
centrifuged supernatants having been removed. Even after stirring,
the precipitate formed at pH 2.0 was partially insoluble. All of
the obtained "precipitate-dissolved solutions" had a pH of around
neutral, which were pH 8.4, pH 8.3, pH 8.3, and pH 8.1.
[0369] The "supernatants of the pH-adjusted culture solutions" at
pH 7.5, pH 4.6, pH 4.0, and pH 2.2, and the "precipitate-dissolved
solutions" obtained by the above-described operations were
subjected to reducing SDS-PAGE, and bands of PIns were checked to
evaluate the precipitation and solubilization of PIns due to the pH
change.
[0370] The recovery ratios of PIns from the "pH-adjusted culture
solutions" at pH 7.5, pH 4.6, pH 4.0, and pH 2.2 were respectively
7%, 6%, 0%, and 7%. FIG. 4-A shows the relation between the
calculated recovery ratio and the pH of the "pH-adjusted culture
solutions."
[0371] FIG. 4-B shows the pH of the "pH-adjusted culture
solutions," band portions of the fusion protein PIns in images of
electrophoresis of the "supernatants of the pH-adjusted culture
solutions" and the corresponding "precipitate-dissolved solutions,"
and the recovery ratio.
[0372] The PIns was present in the "supernatant of the pH-adjusted
culture solution" at pH 7.5 and also detected in the "supernatants
of the pH-adjusted culture solutions" at pH 4.6, pH 4.0, and pH
2.2, but not detected in the "precipitate-dissolved solutions."
Example 4
Production of Fusion Protein Having Proinsulin
[0373] In Example 4, proinsulin (86 amino acid residues) (PIns) was
used as a target protein, a sequence consisting of 250 amino acid
residues from the N-terminus of a CspB mature protein (CspB250) was
used as a protein having a self-assembly capability, and a
coryneform bacterium C. glutamicum was used as a host.
(1) Secretory Expression of Fusion Protein CspB250TEV-Proinsulin
(Abbreviated as 250-PIns) Using pPKK250PIns
[0374] Using the plasmid pPKK250PIns described in Example 1 (1)
(ii), a C. glutamicum YDK010 strain described in WO01/23591 was
transformed. The obtained transformed strain was cultured at
30.degree. C. for 72 hours in MM liquid media (120 g of glucose,
0.4 g of magnesium sulfate heptahydrate, 30 g of ammonium sulfate,
1 g of potassium dihydrogen phosphate, 0.01 g of iron sulfate
heptahydrate, 0.01 g of manganese sulfate pentahydrate, 200 .mu.g
of thiamine hydrochloride, 500 .mu.g of biotin, 0.15 g of
DL-methionine, 50 g of calcium carbonate, adjusted to 1 L with
water and to pH 7.5) containing 25 mg/l of kanamycin. After the
culturing was completed, each culture solution was centrifuged. The
resulting culture supernatant was subjected to reducing SDS-PAGE.
Staining with CBB R-250 (manufactured by Bio-Rad Laboratories,
Inc.) showed a band of the target protein.
(2) Removal of Microbial Cells from Culture Solution, and Storage
of Microbial Cell-Removed Culture Solution
[0375] carried out in the same manner as in Example 1.
(3) Precipitation and Solubilization of Fusion Protein 250-PIns due
to pH Change
[0376] The cryopreserved microbial cell-removed culture solution
was thawed at 25.degree. C. and centrifuged using a centrifuge at
12000 G for 1 minute to separate a precipitate formed during the
cryopreservation. To four microtubes, 0, 5, 10, and 30 .mu.L of
aqueous solutions of 0.5 M sulfuric acid and 30, 25, 20, and 0
.mu.L of Milli Q water were respectively added and uniformly mixed.
Then, 100 .mu.L of the resulting centrifuged supernatant was
dispensed into each of the tubes. All of the mixtures were adjusted
to a volume of 130 .mu.L.
[0377] After stirring, these were left alone for 10 minutes.
Thereby, "pH-adjusted culture solutions" at pH 7.8, pH 4.4, pH 3.0,
and pH 1.7 were obtained (corresponding to "solution obtained in
step (2)"). The "pH-adjusted culture solutions" were not clouded,
and no precipitation formation was observed visually. Those
"pH-adjusted culture solutions" were centrifuged using a centrifuge
at 12000 G for 5 minute. As a result, precipitates (corresponding
to "solid separated in step (3)") were observed in the "pH-adjusted
culture solutions" at pH 4.4 or lower. After these precipitates
formed by the pH change were centrifuged, the resulting centrifuged
supernatants were transferred as "supernatants of the pH-adjusted
culture solutions" (corresponding to "solution after solid
separation in step (3)") to different microtubes separately. To the
remaining precipitates, a buffer (100 mM Tris-HCl, pH 8.5) was
added in the same volume as those of the centrifuged supernatants
having been removed. By stirring, the precipitates were immediately
dissolved, and "precipitate-dissolved solutions" were obtained
(corresponding to "solution obtained in step (4)"). All of the
"precipitate-dissolved solutions" had a pH of around neutral, pH
8.3.
[0378] The "supernatants of the pH-adjusted culture solutions" at
pH 7.8, pH 4.4, pH 3.0, and pH 1.7, and the "precipitate-dissolved
solutions" obtained by the above-described operations were
subjected to reducing SDS-PAGE, and bands of 250-PIns were checked
to evaluate the precipitation and solubilization of 250-PIns due to
the pH change.
[0379] The calculated recovery ratios of 250-PIns from the
"pH-adjusted culture solutions" at pH 7.8, pH 4.4, pH 3.0, and pH
1.7 were respectively 7%, 66%, 70%, and 74%. FIG. 5-A shows the
relation between the calculated recovery ratio and the pH of the
"pH-adjusted culture solutions."
[0380] FIG. 5-B shows the pH of the "pH-adjusted culture
solutions," band portions of the fusion protein 250-PIns in images
of electrophoresis of the "supernatants of the pH-adjusted culture
solutions" and the corresponding "precipitate-dissolved solutions,"
and the recovery ratio.
[0381] The 250-PIns was present in the "supernatant of the
pH-adjusted culture solution" at pH 7.8. Weak bands thereof were
detected in the "supernatants of the pH-adjusted culture solutions"
at pH 4.4, pH 3.0, and pH 1.7, while strong bands were detected in
the "precipitate-dissolved solutions" at these pHs.
Example 5
Production of Fusion Protein Having Proinsulin
[0382] In Example 5, proinsulin (86 amino acid residues) (PIns) was
used as a target protein, a sequence consisting of 17 amino acid
residues from the N-terminus of a CspB mature protein (CspB17) was
used as a protein having a self-assembly capability, and a
coryneform bacterium C. glutamicum was used as a host.
(1) Secretory Expression of Fusion Protein CspB17TEV-Proinsulin
(Abbreviated as 17-PIns) Using pPKK17PIns
[0383] Using the plasmid pPKK17PIns described in Example 1 (1)
(ii), a C. glutamicum YDK010 strain described in WO01/23591 was
transformed. The obtained transformed strain was cultured at
30.degree. C. for 72 hours in MM liquid media (120 g of glucose,
0.4 g of magnesium sulfate heptahydrate, 30 g of ammonium sulfate,
1 g of potassium dihydrogen phosphate, 0.01 g of iron sulfate
heptahydrate, 0.01 g of manganese sulfate pentahydrate, 200 .mu.g
of thiamine hydrochloride, 500 .mu.g of biotin, 0.15 g of
DL-methionine, 50 g of calcium carbonate, adjusted to 1 L with
water and to pH 7.5) containing 25 mg/l of kanamycin. After the
culturing was completed, each culture solution was centrifuged. The
resulting culture supernatant was subjected to reducing SDS-PAGE.
Staining with CBB R-250 (manufactured by Bio-Rad Laboratories,
Inc.) showed a band of the target protein.
(2) Removal of Microbial Cells from Culture Solution, and Storage
of Microbial Cell-Removed Culture Solution
[0384] carried out in the same manner as in Example 1.
(3) Precipitation and Solubilization of Fusion Protein 17-PIns due
to pH Change
[0385] The cryopreserved microbial cell-removed culture solution
was thawed at 25.degree. C. and centrifuged using a centrifuge at
12000 G for 1 minute to separate a precipitate formed during the
cryopreservation. To four microtubes, 0, 5, 10, and 30 .mu.L of
aqueous solutions of 0.5 M sulfuric acid and 30, 25, 20, and 0
.mu.L of Milli Q water were respectively added and uniformly mixed.
Then, 100 .mu.L of the resulting centrifuged supernatant was
dispensed into each of the tubes. All of the mixtures were adjusted
to a volume of 130 .mu.L.
[0386] After stirring, these were left alone for 10 minutes.
Thereby, "pH-adjusted culture solutions" at pH 7.8, pH 4.6, pH 3.7,
and pH 2.0 were obtained (corresponding to "solution obtained in
step (2)"). The "pH-adjusted culture solution" at pH 2.0 was
clouded, and precipitation formation was observed. Those
"pH-adjusted culture solutions" were centrifuged using a centrifuge
at 12000 G for 5 minutes to separate precipitates formed by the pH
change (corresponding to "solid separated in step (3)"). The
resulting centrifuged supernatants were transferred as
"supernatants of the pH-adjusted culture solutions" (corresponding
to "solution after solid separation in step (3)") to different
microtubes separately. To the remaining precipitates, a buffer (100
mM Tris-HCl, pH 8.5) was added in the same volume as those of the
centrifuged supernatants having been removed. By stirring, the
precipitates were immediately dissolved; however, the precipitate
formed at pH 2.0 was partially insoluble. All of the obtained
"precipitate-dissolved solutions" (corresponding to "solution
obtained in step (4)") had a pH of around neutral, pH 8.5.
[0387] The "supernatants of the pH-adjusted culture solutions" at
pH 7.8, pH 4.6, pH 3.7, and pH 2.0, and the "precipitate-dissolved
solutions" obtained by the above-described operations were
subjected to reducing SDS-PAGE, and bands of 17-PIns were checked
to evaluate the precipitation and solubilization of 17-PIns due to
the pH change.
[0388] The recovery ratios of 17-PIns from the "pH-adjusted culture
solutions" at pH 7.8, pH 4.6, pH 3.7, and pH 2.0 were respectively
9%, 46%, 43%, and 45%. FIG. 6-A shows the relation between the
calculated recovery ratio and the pH of the "pH-adjusted culture
solutions."
[0389] FIG. 6-B shows the pH of the "pH-adjusted culture
solutions," band portions of the fusion protein 17-PIns in images
of electrophoresis of the "supernatants of the pH-adjusted culture
solutions" and the corresponding "precipitate-dissolved solutions,"
and the recovery ratio.
[0390] The 17-PIns was present in the "supernatant of the
pH-adjusted culture solution" at pH 7.8, and also detected as
strong bands in the "precipitate-dissolved solutions" at pH 4.6, pH
3.7, and pH 2.0.
Example 6
Production of Fusion Protein Having Proinsulin
[0391] In Example 6, proinsulin (86 amino acid residues) (PIns) was
used as a target protein, a sequence consisting of 6 amino acid
residues from the N-terminus of a CspB mature protein (CspB6) was
used as a protein having a self-assembly capability, and a
coryneform bacterium C. glutamicum was used as a host.
(1) Secretory Expression of Fusion Protein CspB6TEV-Proinsulin
(Abbreviated as 6-PIns) Using pPKK6PIns
[0392] Using the plasmid pPKK6PIns described in Example 1 (1) (ii),
a C. glutamicum YDK010 strain described in WO01/23591 was
transformed. The obtained transformed strain was cultured at
30.degree. C. for 72 hours in MM liquid media (120 g of glucose,
0.4 g of magnesium sulfate heptahydrate, 30 g of ammonium sulfate,
1 g of potassium dihydrogen phosphate, 0.01 g of iron sulfate
heptahydrate, 0.01 g of manganese sulfate pentahydrate, 200 .mu.g
of thiamine hydrochloride, 500 .mu.g of biotin, 0.15 g of
DL-methionine, 50 g of calcium carbonate, adjusted to 1 L with
water and to pH 7.5) containing 25 mg/l of kanamycin. After the
culturing was completed, each culture solution was centrifuged. The
resulting culture supernatant was subjected to reducing SDS-PAGE.
Staining with CBB R-250 (manufactured by Bio-Rad Laboratories,
Inc.) showed a band of the target protein.
(2) Removal of Microbial Cells from Culture Solution, and Storage
of Microbial Cell-Removed Culture Solution
[0393] carried out in the same manner as in Example 1.
(3) Precipitation and Solubilization of Fusion Protein 6-PIns Due
to pH Change
[0394] The cryopreserved microbial cell-removed culture solution
was thawed at 25.degree. C. and centrifuged using a centrifuge at
12000 G for 1 minute to separate a precipitate formed during the
cryopreservation. To four microtubes, 0, 5, 10, and 30 .mu.L of
aqueous solutions of 0.5 M sulfuric acid and 30, 25, 20, and 0
.mu.L of Milli Q water were respectively added and uniformly mixed.
Then, 100 .mu.L of the resulting centrifuged supernatant was
dispensed into each of the tubes. All of the mixtures were adjusted
to a volume of 130 .mu.L.
[0395] After stirring, these were left alone for 10 minutes.
Thereby, "pH-adjusted culture solutions" at pH 7.6, pH 4.6, pH 3.5,
and pH 1.8 were obtained (corresponding to "solution obtained in
step (2)"). The "pH-adjusted culture solution" at pH 1.8 was
clouded, and precipitation formation was observed. Those
"pH-adjusted culture solutions" were centrifuged using a centrifuge
at 12000 G for 5 minutes to separate precipitates formed by the pH
change (corresponding to "solid separated in step (3)"). The
resulting centrifuged supernatants were transferred as
"supernatants of the pH-adjusted culture solutions" (corresponding
to "solution after solid separation in step (3)") to different
microtubes separately. To the remaining precipitates, a buffer (100
mM Tris-HCl, pH 8.5) was added in the same volume as those of the
centrifuged supernatants having been removed. By stirring, the
precipitates were immediately dissolved; however, the precipitate
formed at pH 1.8 was partially insoluble. All of the obtained
"precipitate-dissolved solutions" (corresponding to "solution
obtained in step (4)") had a pH of around neutral, pH 8.5.
[0396] The "supernatants of the pH-adjusted culture solutions" at
pH 7.6, pH 4.6, pH 3.5, and pH 1.8, and the "precipitate-dissolved
solutions" obtained by the above-described operations were
subjected to reducing SDS-PAGE, and bands of 6-PIns were checked to
evaluate the precipitation and solubilization of 6-PIns due to the
pH change.
[0397] The calculated recovery ratios of 6-PIns from the
"pH-adjusted culture solutions" at pH 7.6, pH 4.6, pH 3.5, and pH
1.8 were respectively 3%, 34%, 31%, and 45%. FIG. 7-A shows the
relation between the calculated recovery ratio and the pH of the
"pH-adjusted culture solutions."
[0398] FIG. 7-B shows the pH of the "pH-adjusted culture
solutions," band portions of the fusion protein 6-PIns in images of
electrophoresis of the "supernatants of the pH-adjusted culture
solutions" and the corresponding "precipitate-dissolved solutions,"
and the recovery ratio.
[0399] The 6-PIns was present in the "supernatant of the
pH-adjusted culture solution" at pH 7.6, and also detected as bands
in the "precipitate-dissolved solutions" at pH 4.6, pH 3.5, and pH
1.8.
Example 7
Production of Fusion Protein Having Teriparatide
[0400] In Example 7, in the same manner as in Example 1, a
bioactive peptide teriparatide (34 amino acid residues) (Teri) was
used as a target protein, a sequence consisting of 50 amino acid
residues from the N-terminus of a CspB mature protein (CspB50) was
used as a protein having a self-assembly capability, a proTEV
protease recognition sequence was used as an amino acid sequence
used for an enzymatic cleavage, and a coryneform bacterium C.
glutamicum was used as a host.
[0401] Nevertheless, hydrochloric acid was used to adjust the pH of
the solution containing the fusion protein (in Example 1, sulfuric
acid was used).
(1) Construction of Teriparatide-Secretory Expression Plasmid
(pPKK50TEV-Teri)
[0402] carried out in the same manner as in Example 1.
[0403] (2) Secretory Expression of Fusion Protein Using
pPKK50TEV-Teri
[0404] carried out in the same manner as in Example 1.
(3) Removal of Microbial Cells from Culture Solution, and Storage
of Microbial Cell-Removed Culture Solution
[0405] the same as in Example 1.
(4) Precipitation and Solubilization of Fusion Protein (Made of
CspB50 and Teri (50-Teri)) Due to pH Change
[0406] The cryopreserved microbial cell-removed culture solution
was thawed at 25.degree. C. and centrifuged using a centrifuge at
12000 G for 1 minute to separate a precipitate formed during the
cryopreservation. To four microtubes, 0, 3.5, 5, and 10 .mu.L of
aqueous solutions of 1 M hydrochloric acid and 10, 6.5, 5, and 0
.mu.L of Milli Q water were respectively added and uniformly mixed.
Then, 100 .mu.L of the resulting centrifuged supernatant was
dispensed into each of the tubes. All of the mixtures were adjusted
to a volume of 110 .mu.L.
[0407] After stirring, these were left alone for 10 minutes.
Thereby, "pH-adjusted culture solutions" at pH 7.9, pH 7.0, pH 5.3,
and pH 3.1 were obtained (corresponding to "solution obtained in
step (2)"). The "pH-adjusted culture solutions" at pH 7.0 or lower
were clouded, and precipitation formation was observed. Those
"pH-adjusted culture solutions" were centrifuged using a centrifuge
at 12000 G for 5 minutes to separate precipitates formed by the pH
change (corresponding to "solid separated in step (3)"). The
resulting centrifuged supernatants were transferred as
"supernatants of the pH-adjusted culture solutions" (corresponding
to "solution after solid separation in step (3)") to different
microtubes separately. To the remaining precipitates, a buffer (100
mM Tris-HCl, pH 8.5) was added in the same volume as those of the
centrifuged supernatants having been removed. By stirring, the
precipitates were immediately dissolved, and "precipitate-dissolved
solutions" were obtained (corresponding to "solution obtained in
step (4)"). All of the "precipitate-dissolved solutions" had a pH
of around neutral, which were pH 8.5, pH 8.5, pH 8.4, and pH
8.3.
[0408] The "supernatants of the pH-adjusted culture solutions" at
pH 7.9, pH 7.0, pH 5.3, and pH 3.1, and the "precipitate-dissolved
solutions" obtained by the above-described operations were
subjected to reducing SDS-PAGE and bands of 50-Teri were checked to
evaluate the precipitation and solubilization of 50-Teri due to the
pH change.
[0409] The calculated recovery ratios of 50-Teri from the
"pH-adjusted culture solutions" at pH 7.9, pH 7.0, pH 5.3, and pH
3.1 were respectively 2%, 80%, 100%, and 99%. FIG. 8-A shows the
relation between the calculated recovery ratio and the pH of the
"pH-adjusted culture solutions."
[0410] FIG. 8-B shows the pH of the "pH-adjusted culture
solutions," band portions of the fusion protein 50-Teri in images
of electrophoresis of the "supernatants of the pH-adjusted culture
solutions" and the corresponding "precipitate-dissolved solutions,"
and the recovery ratio.
[0411] The 50-Teri was present in the "supernatant of the
pH-adjusted culture solution" at pH 7.9. The 50-Teri was not
detected in the "supernatants of the pH-adjusted culture solutions"
at pH 7.0, pH 5.3, and pH 3.1, but was detected in the
corresponding "precipitate-dissolved solutions" at these pHs.
Example 8
Production of Fusion Protein Having Teriparatide
[0412] In Example 8, in the same manner as in Example 1, a
bioactive peptide teriparatide (34 amino acid residues) (Teri) was
used as a target protein, a sequence consisting of 50 amino acid
residues from the N-terminus of a CspB mature protein (CspB50) was
used as a protein having a self-assembly capability, a proTEV
protease recognition sequence was used as an amino acid sequence
used for an enzymatic cleavage, and a coryneform bacterium C.
glutamicum was used as a host.
[0413] Nevertheless, acetic acid was used to adjust the pH of the
solution containing the fusion protein (in Example 1, sulfuric acid
was used).
(1) Construction of Teriparatide-Secretory Expression Plasmid
(pPKK50TEV-Teri)
[0414] carried out in the same manner as in Example 1.
(2) Secretory Expression of Fusion Protein CspB50TEV-Teriparatide
(abbreviated as 50-Teri) Using pPKK50TEV-Teri
[0415] carried out in the same manner as in Example 1.
(3) Removal of Microbial Cells from Culture Solution, and Storage
of Microbial Cell-Removed Culture Solution
[0416] carried out in the same manner as in Example 1.
(4) Precipitation and Solubilization of Fusion Protein 50-Teri due
to pH Change
[0417] The cryopreserved microbial cell-removed culture solution
was thawed at 25.degree. C. and centrifuged using a centrifuge at
12000 G for 1 minute to separate a precipitate formed during the
cryopreservation. To four microtubes, 0, 2, 4, and 10 .mu.L of
aqueous solutions of 10% acetic acid and 10, 8, 6, and 0 .mu.L of
Milli Q water were respectively added and uniformly mixed. Then,
100 .mu.L of the resulting centrifuged supernatant was dispensed
into each of the tubes. All of the mixtures were adjusted to a
volume of 110 .mu.L.
[0418] After stirring, these were left alone for 10 minutes.
Thereby, "pH-adjusted culture solutions" at pH 7.9, pH 6.8, pH 5.0,
and pH 4.3 were obtained (corresponding to "solution obtained in
step (2)"). The "pH-adjusted culture solutions" at pH 6.8 or lower
were clouded, and precipitation formation was observed. Those
"pH-adjusted culture solutions" were centrifuged using a centrifuge
at 12000 G for 5 minutes to separate precipitates formed by the pH
change (corresponding to "solid separated in step (3)"). The
resulting centrifuged supernatants were transferred as
"supernatants of the pH-adjusted culture solutions" (corresponding
to "solution after solid separation in step (3)") to different
microtubes separately. To the remaining precipitates, a buffer (100
mM Tris-HCl, pH 8.5) was added in the same volume as those of the
centrifuged supernatants having been removed. By stirring, the
precipitates were immediately dissolved, and "precipitate-dissolved
solutions" were obtained (corresponding to "solution obtained in
step (4)"). All of the "precipitate-dissolved solutions" had a pH
of around neutral, which were pH 8.5, pH 8.3, pH 8.1, and pH
7.9.
[0419] The "supernatants of the pH-adjusted culture solutions" at
pH 7.9, pH 6.8, pH 5.0, and pH 4.3, and the "precipitate-dissolved
solutions" obtained by the above-described operations were
subjected to reducing SDS-PAGE, and bands of 50-Teri were checked
to evaluate the precipitation and solubilization of 50-Teri due to
the pH change.
[0420] The recovery ratios from the "pH-adjusted culture solutions"
at pH 7.9, pH 6.8, pH 5.0, and pH 4.3 were respectively 2%, 95%,
99%, and 98%. FIG. 9-A shows the relation between the calculated
recovery ratio and the pH of the "pH-adjusted culture
solutions."
[0421] FIG. 9-B shows the pH of the "pH-adjusted culture
solutions," band portions of the fusion protein 50-Teri in images
of electrophoresis of the "supernatants of the pH-adjusted culture
solutions" and the corresponding "precipitate-dissolved solutions,"
and the recovery ratio.
[0422] The 50-Teri was present in the "supernatant of the
pH-adjusted culture solution" at pH 7.9. The 50-Teri was not
detected in the "supernatants of the pH-adjusted culture solutions"
at pH 6.8, pH 5.0, and pH 4.3, but was detected in the
corresponding "precipitate-dissolved solutions" at these pHs.
Example 9
Production of Fusion Protein Having Teriparatide
[0423] In Example 9, in the same manner as in Example 1 and Example
1-2, a bioactive peptide teriparatide (34 amino acid residues)
(Teri) was used as a target protein, a sequence consisting of 50
amino acid residues from the N-terminus of a CspB mature protein
(CspB50) was used as a protein having a self-assembly capability, a
proTEV protease recognition sequence was used as an amino acid
sequence used for an enzymatic cleavage, and a coryneform bacterium
C. glutamicum was used as a host.
(1) Construction of Teriparatide-Secretory Expression Plasmid
(pPKK50TEV-Teri)
[0424] carried out in the same manner as in Example 1.
(2) Secretory Expression of Fusion Protein CspB50TEV-Teriparatide
(abbreviated as 50-Teri) Using pPKK50TEV-Teri
[0425] carried out in the same manner as in Example 1.
[0426] (3) Removal of Microbial Cells from Culture Solution, and
Storage of Microbial Cell-Removed Culture Solution
[0427] carried out in the same manner as in Example 1.
(4) Precipitation and Solubilization of Fusion Protein 50-Teri Due
to pH Change and Evaluation of an Improvement of the Purity of
50-Teri
[0428] The cryopreserved microbial cell-removed culture solution
was thawed at 25.degree. C. and centrifuged using a centrifuge at
12000 G for 1 minute to separate a precipitate formed during the
cryopreservation. To microtube, 50 .mu.L of aqueous solutions of
0.5 M sulfuric acid and 600 .mu.L of the resulting centrifuged
supernatant were added.
[0429] After stirring, these were left alone for 10 minutes.
Thereby, "pH-adjusted culture solution" at pH 3.7 was obtained
(corresponding to "solution obtained in step (2)"). The pH value of
3.7 was used as "such a pH that a recovery ratio is 10% or more"
based on the results of Example 1 and Example 1-2. The "pH-adjusted
culture solution" was centrifuged using a centrifuge at 12000 G for
5 minutes to separate a precipitate formed by the pH change
(corresponding to "solid separated in step (3)"). The resulting
centrifuged supernatant was transferred as "supernatant of the
pH-adjusted culture solution" (corresponding to "solution after
solid separation in step (3)") to a different microtube. To the
remaining precipitate, a buffer (100 mM Tris-HCl, pH 8.5) was added
in the same volume as those of the centrifuged supernatant having
been removed. By stirring, the precipitate was immediately
dissolved, and "precipitate-dissolved solution" was obtained
(corresponding to "solution obtained in step (4)"). The
"precipitate-dissolved solution" had a pH of around neutral, which
was pH 7.4. The pH value of 7.4 was used as "pH of 12 or below but
higher than the pH of the solution obtained in step (2) by 0.1 or
more" based on the results of Example 1 and Example 1-2.
[0430] The microbial cell-removed culture solution (before the
above operations) and the "precipitate-dissolved solution" obtained
by the above-described operations were subjected to reversed-phase
HPLC to determine the peak areas of 50-Teri and so on and calculate
the purity of 50-Teri in each solution according to the following
equation. The calculated purities were compared.
the purity (%)=(the peak area of 50-Teri/total peak
area).times.100.
[0431] The conditions of the reversed-phase HPLC are shown
below.
System: a set of Waters Alliance PDA system Column: YMC-Pack C8
.phi.4.6.times.100 mm, a particle diameter of 5 .mu.m, a pore
diameter of 30 nm Column temp.: 30.degree. C. Mobile phase A: 10 mM
ammonium acetate, 10% acetonitrile, pH 7.0 (non-adjusted pH value)
Mobile phase B: 10 mM ammonium acetate, 80% acetonitrile, pH 7.0
(non-adjusted pH value) Flow rate: 1.0 mL/min
Detection: 280 nm, 220 nm
[0432] Injection volume: 30 .mu.L
TABLE-US-00006 Gradient: Time (min) A (%) B (%) 0 100 0 5 74 26 25
64 36 30 0 100
[0433] Results of reversed-phase HPLC for each solution are shown
in FIG. 10-A and FIG. 10-B. FIG. 10-A shows peaks detected at the
wavelength of 280 nm. FIG. 10-B shows peaks detected at the
wavelength of 220 nm.
[0434] When detected at the wavelength of 280 nm, the purity of
50-Teri in the microbial cell-removed culture solution was 11%
while the purity of 50-Teri in the precipitate-dissolved solution
was improved to 46% (FIG. 10-A). Similarly, when detected at the
wavelength of 220 nm, the purity of 50-Teri in the microbial
cell-removed culture solution was 46% while the purity of 50-Teri
in the precipitate-dissolved solution was improved to 73% (FIG.
10-B).
[0435] This revealed that the above operations according to the
present invention can achieve the improvement of the purity of
50-Teri as well as the recovery of 50-Teri.
INDUSTRIAL APPLICABILITY
[0436] The present invention can be utilized in methods for
producing target proteins.
SEQUENCE LISTING FREE TEXT
[0437] SEQ ID NO: 1: base sequence of cspB gene of C. glutamicum
ATCC13869 [0438] SEQ ID NO: 2: amino acid sequence of CspB protein
of C. glutamicum ATCC13869 [0439] SEQ ID NO: 3: amino acid sequence
of CspB mature protein of C. glutamicum ATCC13869 [0440] SEQ ID NO:
4: amino acid sequence of signal peptide of C. glutamicum-derived
PS1 [0441] SEQ ID NO: 5: amino acid sequence of signal peptide of
C. glutamicum-derived PS2 (CspB) [0442] SEQ ID NO: 6: amino acid
sequence of signal peptide of C. ammoniagenes-derived SlpA (CspA)
[0443] SEQ ID NO: 7: Factor Xa protease recognition sequence [0444]
SEQ ID NO: 8: ProTEV protease recognition sequence [0445] SEQ ID
NOs: 9 to 16: base sequences of DNA for proinsulin total synthesis
[0446] SEQ ID NOs: 17, 18: primers [0447] SEQ ID NO: 19: base
sequence of proinsulin gene [0448] SEQ ID NOs: 20 to 55: primers
[0449] SEQ ID NOs: 56 to 69: base sequences of DNA for human growth
hormone hGH total synthesis [0450] SEQ ID NOs: 70, 71: primers
[0451] SEQ ID NO: 72: base sequence of hGH gene [0452] SEQ ID NOs:
73 to 77: primers [0453] SEQ ID NOs: 78, 79: base sequences of DNA
for teriparatide synthesis [0454] SEQ ID NOs: 80, 81: primers
[0455] SEQ ID NO: 82: base sequence of teriparatide gene [0456] SEQ
ID NOs: 83 to 86: primers [0457] SEQ ID NOs: 87 to 89: primers
[0458] SEQ ID NO: 90: base sequence of DNA for Biva18 synthesis
[0459] SEQ ID NOs: 91, 92: primers [0460] SEQ ID NO: 93: amino acid
sequence of Biva18
Sequence CWU 1
1
9311500DNACorynebacterium glutamicum ATCC13869CDS(1)..(1500) 1atg
ttt aac aac cgt atc cgc act gca gct ctc gct ggt gca atc gca 48Met
Phe Asn Asn Arg Ile Arg Thr Ala Ala Leu Ala Gly Ala Ile Ala 1 5 10
15 atc tcc acc gca gct tcc ggc gta gct atc cca gca ttc gct cag gag
96Ile Ser Thr Ala Ala Ser Gly Val Ala Ile Pro Ala Phe Ala Gln Glu
20 25 30 acc aac cca acc ttc aac atc aac aac ggc ttc aac gat gct
gat gga 144Thr Asn Pro Thr Phe Asn Ile Asn Asn Gly Phe Asn Asp Ala
Asp Gly 35 40 45 tcc acc atc cag cca gtt gag cca gtt aac cac acc
gag gaa acc ctc 192Ser Thr Ile Gln Pro Val Glu Pro Val Asn His Thr
Glu Glu Thr Leu 50 55 60 cgc gac ctg act gac tcc acc ggc gct tac
ctg gaa gag ttc cag tac 240Arg Asp Leu Thr Asp Ser Thr Gly Ala Tyr
Leu Glu Glu Phe Gln Tyr 65 70 75 80 ggc aac gtt gag gaa atc gtt gaa
gca tac ctg cag gtt cag gct tcc 288Gly Asn Val Glu Glu Ile Val Glu
Ala Tyr Leu Gln Val Gln Ala Ser 85 90 95 gca gac gga ttc gat cct
tct gag cag gct gct tac gag gct ttc gag 336Ala Asp Gly Phe Asp Pro
Ser Glu Gln Ala Ala Tyr Glu Ala Phe Glu 100 105 110 gct gct cgc gtt
cgt gca tcc cag gag ctc gcg gct tcc gct gag acc 384Ala Ala Arg Val
Arg Ala Ser Gln Glu Leu Ala Ala Ser Ala Glu Thr 115 120 125 atc act
aag acc cgc gag tcc gtt gct tac gca ctc aag gct gac cgc 432Ile Thr
Lys Thr Arg Glu Ser Val Ala Tyr Ala Leu Lys Ala Asp Arg 130 135 140
gaa gct acc gca gct ttc gag gct tac ctc agc gct ctt cgt cag gtt
480Glu Ala Thr Ala Ala Phe Glu Ala Tyr Leu Ser Ala Leu Arg Gln Val
145 150 155 160 tca gtc atc aac gat ctg atc gct gat gct aac gcc aag
aac aag act 528Ser Val Ile Asn Asp Leu Ile Ala Asp Ala Asn Ala Lys
Asn Lys Thr 165 170 175 gac ttt gca gag atc gag ctc tac gat gtt ctt
tac acc gac gcc gac 576Asp Phe Ala Glu Ile Glu Leu Tyr Asp Val Leu
Tyr Thr Asp Ala Asp 180 185 190 atc tct ggc gat gct cca ctt ctt gct
cct gca tac aag gag ctg aag 624Ile Ser Gly Asp Ala Pro Leu Leu Ala
Pro Ala Tyr Lys Glu Leu Lys 195 200 205 gac ctt cag gct gag gtt gac
gca gac ttc gag tgg ttg ggc gag ttc 672Asp Leu Gln Ala Glu Val Asp
Ala Asp Phe Glu Trp Leu Gly Glu Phe 210 215 220 gca att gat aac aat
gaa gac aac tac gtc att cgt act cac atc cct 720Ala Ile Asp Asn Asn
Glu Asp Asn Tyr Val Ile Arg Thr His Ile Pro 225 230 235 240 gct gta
gag gca ctc aag gca gcg atc gat tca ctg gtc gac acc gtt 768Ala Val
Glu Ala Leu Lys Ala Ala Ile Asp Ser Leu Val Asp Thr Val 245 250 255
gag cca ctt cgt gca gac gct atc gct aag aac atc gag gct cag aag
816Glu Pro Leu Arg Ala Asp Ala Ile Ala Lys Asn Ile Glu Ala Gln Lys
260 265 270 tct gac gtt ctg gtt ccc cag ctc ttc ctc gag cgt gca act
gca cag 864Ser Asp Val Leu Val Pro Gln Leu Phe Leu Glu Arg Ala Thr
Ala Gln 275 280 285 cgc gac acc ctg cgt gtt gta gag gca atc ttc tct
acc tct gct cgt 912Arg Asp Thr Leu Arg Val Val Glu Ala Ile Phe Ser
Thr Ser Ala Arg 290 295 300 tac gtt gaa ctc tac gag aac gtc gag aac
gtt aac gtt gag aac aag 960Tyr Val Glu Leu Tyr Glu Asn Val Glu Asn
Val Asn Val Glu Asn Lys 305 310 315 320 acc ctt cgc cag cac tac tct
tcc ctg atc cct aac ctc ttc atc gca 1008Thr Leu Arg Gln His Tyr Ser
Ser Leu Ile Pro Asn Leu Phe Ile Ala 325 330 335 gcg gtt ggc aac atc
aac gag ctc aac aat gca gat cag gct gca cgt 1056Ala Val Gly Asn Ile
Asn Glu Leu Asn Asn Ala Asp Gln Ala Ala Arg 340 345 350 gag ctc ttc
ctc gat tgg gac acc gac ctc acc acc aac gat gag gac 1104Glu Leu Phe
Leu Asp Trp Asp Thr Asp Leu Thr Thr Asn Asp Glu Asp 355 360 365 gaa
gct tac tac cag gct aag ctc gac ttc gct atc gag acc tac gca 1152Glu
Ala Tyr Tyr Gln Ala Lys Leu Asp Phe Ala Ile Glu Thr Tyr Ala 370 375
380 aag atc ctg atc aac ggt gaa gtt tgg cag gag cca ctc gct tac gtc
1200Lys Ile Leu Ile Asn Gly Glu Val Trp Gln Glu Pro Leu Ala Tyr Val
385 390 395 400 cag aac ctg gat gca ggc gca cgt cag gaa gca gct gac
cgc gaa gca 1248Gln Asn Leu Asp Ala Gly Ala Arg Gln Glu Ala Ala Asp
Arg Glu Ala 405 410 415 gag cgc gca gct gac gca gca tac cgc gct gag
cag ctc cgc atc gct 1296Glu Arg Ala Ala Asp Ala Ala Tyr Arg Ala Glu
Gln Leu Arg Ile Ala 420 425 430 cag gaa gca gct gac gct cag aag gct
ctc gct gag gct ctt gct aat 1344Gln Glu Ala Ala Asp Ala Gln Lys Ala
Leu Ala Glu Ala Leu Ala Asn 435 440 445 gca ggc aac aac gac aac ggt
ggc gac aac tcc tcc gac gac aag gga 1392Ala Gly Asn Asn Asp Asn Gly
Gly Asp Asn Ser Ser Asp Asp Lys Gly 450 455 460 acc ggt tct tcc gac
atc gga acc tgg gga cct ttc gca gca att gca 1440Thr Gly Ser Ser Asp
Ile Gly Thr Trp Gly Pro Phe Ala Ala Ile Ala 465 470 475 480 gct atc
atc gca gca atc gca gct atc ttc cca ttc ctc tcc ggt atc 1488Ala Ile
Ile Ala Ala Ile Ala Ala Ile Phe Pro Phe Leu Ser Gly Ile 485 490 495
gtt aag ttc taa 1500Val Lys Phe 2499PRTCorynebacterium glutamicum
ATCC13869 2Met Phe Asn Asn Arg Ile Arg Thr Ala Ala Leu Ala Gly Ala
Ile Ala 1 5 10 15 Ile Ser Thr Ala Ala Ser Gly Val Ala Ile Pro Ala
Phe Ala Gln Glu 20 25 30 Thr Asn Pro Thr Phe Asn Ile Asn Asn Gly
Phe Asn Asp Ala Asp Gly 35 40 45 Ser Thr Ile Gln Pro Val Glu Pro
Val Asn His Thr Glu Glu Thr Leu 50 55 60 Arg Asp Leu Thr Asp Ser
Thr Gly Ala Tyr Leu Glu Glu Phe Gln Tyr 65 70 75 80 Gly Asn Val Glu
Glu Ile Val Glu Ala Tyr Leu Gln Val Gln Ala Ser 85 90 95 Ala Asp
Gly Phe Asp Pro Ser Glu Gln Ala Ala Tyr Glu Ala Phe Glu 100 105 110
Ala Ala Arg Val Arg Ala Ser Gln Glu Leu Ala Ala Ser Ala Glu Thr 115
120 125 Ile Thr Lys Thr Arg Glu Ser Val Ala Tyr Ala Leu Lys Ala Asp
Arg 130 135 140 Glu Ala Thr Ala Ala Phe Glu Ala Tyr Leu Ser Ala Leu
Arg Gln Val 145 150 155 160 Ser Val Ile Asn Asp Leu Ile Ala Asp Ala
Asn Ala Lys Asn Lys Thr 165 170 175 Asp Phe Ala Glu Ile Glu Leu Tyr
Asp Val Leu Tyr Thr Asp Ala Asp 180 185 190 Ile Ser Gly Asp Ala Pro
Leu Leu Ala Pro Ala Tyr Lys Glu Leu Lys 195 200 205 Asp Leu Gln Ala
Glu Val Asp Ala Asp Phe Glu Trp Leu Gly Glu Phe 210 215 220 Ala Ile
Asp Asn Asn Glu Asp Asn Tyr Val Ile Arg Thr His Ile Pro 225 230 235
240 Ala Val Glu Ala Leu Lys Ala Ala Ile Asp Ser Leu Val Asp Thr Val
245 250 255 Glu Pro Leu Arg Ala Asp Ala Ile Ala Lys Asn Ile Glu Ala
Gln Lys 260 265 270 Ser Asp Val Leu Val Pro Gln Leu Phe Leu Glu Arg
Ala Thr Ala Gln 275 280 285 Arg Asp Thr Leu Arg Val Val Glu Ala Ile
Phe Ser Thr Ser Ala Arg 290 295 300 Tyr Val Glu Leu Tyr Glu Asn Val
Glu Asn Val Asn Val Glu Asn Lys 305 310 315 320 Thr Leu Arg Gln His
Tyr Ser Ser Leu Ile Pro Asn Leu Phe Ile Ala 325 330 335 Ala Val Gly
Asn Ile Asn Glu Leu Asn Asn Ala Asp Gln Ala Ala Arg 340 345 350 Glu
Leu Phe Leu Asp Trp Asp Thr Asp Leu Thr Thr Asn Asp Glu Asp 355 360
365 Glu Ala Tyr Tyr Gln Ala Lys Leu Asp Phe Ala Ile Glu Thr Tyr Ala
370 375 380 Lys Ile Leu Ile Asn Gly Glu Val Trp Gln Glu Pro Leu Ala
Tyr Val 385 390 395 400 Gln Asn Leu Asp Ala Gly Ala Arg Gln Glu Ala
Ala Asp Arg Glu Ala 405 410 415 Glu Arg Ala Ala Asp Ala Ala Tyr Arg
Ala Glu Gln Leu Arg Ile Ala 420 425 430 Gln Glu Ala Ala Asp Ala Gln
Lys Ala Leu Ala Glu Ala Leu Ala Asn 435 440 445 Ala Gly Asn Asn Asp
Asn Gly Gly Asp Asn Ser Ser Asp Asp Lys Gly 450 455 460 Thr Gly Ser
Ser Asp Ile Gly Thr Trp Gly Pro Phe Ala Ala Ile Ala 465 470 475 480
Ala Ile Ile Ala Ala Ile Ala Ala Ile Phe Pro Phe Leu Ser Gly Ile 485
490 495 Val Lys Phe 3469PRTCorynebacterium glutamicum ATCC13869
3Gln Glu Thr Asn Pro Thr Phe Asn Ile Asn Asn Gly Phe Asn Asp Ala 1
5 10 15 Asp Gly Ser Thr Ile Gln Pro Val Glu Pro Val Asn His Thr Glu
Glu 20 25 30 Thr Leu Arg Asp Leu Thr Asp Ser Thr Gly Ala Tyr Leu
Glu Glu Phe 35 40 45 Gln Tyr Gly Asn Val Glu Glu Ile Val Glu Ala
Tyr Leu Gln Val Gln 50 55 60 Ala Ser Ala Asp Gly Phe Asp Pro Ser
Glu Gln Ala Ala Tyr Glu Ala 65 70 75 80 Phe Glu Ala Ala Arg Val Arg
Ala Ser Gln Glu Leu Ala Ala Ser Ala 85 90 95 Glu Thr Ile Thr Lys
Thr Arg Glu Ser Val Ala Tyr Ala Leu Lys Ala 100 105 110 Asp Arg Glu
Ala Thr Ala Ala Phe Glu Ala Tyr Leu Ser Ala Leu Arg 115 120 125 Gln
Val Ser Val Ile Asn Asp Leu Ile Ala Asp Ala Asn Ala Lys Asn 130 135
140 Lys Thr Asp Phe Ala Glu Ile Glu Leu Tyr Asp Val Leu Tyr Thr Asp
145 150 155 160 Ala Asp Ile Ser Gly Asp Ala Pro Leu Leu Ala Pro Ala
Tyr Lys Glu 165 170 175 Leu Lys Asp Leu Gln Ala Glu Val Asp Ala Asp
Phe Glu Trp Leu Gly 180 185 190 Glu Phe Ala Ile Asp Asn Asn Glu Asp
Asn Tyr Val Ile Arg Thr His 195 200 205 Ile Pro Ala Val Glu Ala Leu
Lys Ala Ala Ile Asp Ser Leu Val Asp 210 215 220 Thr Val Glu Pro Leu
Arg Ala Asp Ala Ile Ala Lys Asn Ile Glu Ala 225 230 235 240 Gln Lys
Ser Asp Val Leu Val Pro Gln Leu Phe Leu Glu Arg Ala Thr 245 250 255
Ala Gln Arg Asp Thr Leu Arg Val Val Glu Ala Ile Phe Ser Thr Ser 260
265 270 Ala Arg Tyr Val Glu Leu Tyr Glu Asn Val Glu Asn Val Asn Val
Glu 275 280 285 Asn Lys Thr Leu Arg Gln His Tyr Ser Ser Leu Ile Pro
Asn Leu Phe 290 295 300 Ile Ala Ala Val Gly Asn Ile Asn Glu Leu Asn
Asn Ala Asp Gln Ala 305 310 315 320 Ala Arg Glu Leu Phe Leu Asp Trp
Asp Thr Asp Leu Thr Thr Asn Asp 325 330 335 Glu Asp Glu Ala Tyr Tyr
Gln Ala Lys Leu Asp Phe Ala Ile Glu Thr 340 345 350 Tyr Ala Lys Ile
Leu Ile Asn Gly Glu Val Trp Gln Glu Pro Leu Ala 355 360 365 Tyr Val
Gln Asn Leu Asp Ala Gly Ala Arg Gln Glu Ala Ala Asp Arg 370 375 380
Glu Ala Glu Arg Ala Ala Asp Ala Ala Tyr Arg Ala Glu Gln Leu Arg 385
390 395 400 Ile Ala Gln Glu Ala Ala Asp Ala Gln Lys Ala Leu Ala Glu
Ala Leu 405 410 415 Ala Asn Ala Gly Asn Asn Asp Asn Gly Gly Asp Asn
Ser Ser Asp Asp 420 425 430 Lys Gly Thr Gly Ser Ser Asp Ile Gly Thr
Trp Gly Pro Phe Ala Ala 435 440 445 Ile Ala Ala Ile Ile Ala Ala Ile
Ala Ala Ile Phe Pro Phe Leu Ser 450 455 460 Gly Ile Val Lys Phe 465
443PRTCorynebacterium glutamicum 4Met Arg Asp Thr Ala Phe Arg Ser
Ile Lys Ala Lys Ala Gln Ala Lys 1 5 10 15 Arg Arg Ser Leu Trp Ile
Ala Ala Gly Ala Val Pro Thr Ala Ile Ala 20 25 30 Leu Thr Met Ser
Leu Ala Pro Met Ala Ser Ala 35 40 530PRTCorynebacterium glutamicum
5Met Phe Asn Asn Arg Ile Arg Thr Ala Ala Leu Ala Gly Ala Ile Ala 1
5 10 15 Ile Ser Thr Ala Ala Ser Gly Val Ala Ile Pro Ala Phe Ala 20
25 30 625PRTCorynebacterium ammoniagenes 6Met Lys Arg Met Lys Ser
Leu Ala Ala Ala Leu Thr Val Ala Gly Ala 1 5 10 15 Met Leu Ala Ala
Pro Val Ala Thr Ala 20 25 74PRTArtificial SequenceFactorXa 7Ile Glu
Gly Arg 1 86PRTArtificial SequenceProTEV 8Glu Asn Leu Tyr Phe Gln 1
5 960DNAArtificial SequencePIns 9atggcgctct ggatgcgcct gctgccactc
ctggcgctcc tggcactgtg gggaccagat 601060DNAArtificial SequencePIns
10gggagccgca aagatgttgg ttcacgaagg cggcagcagg atctggtccc cacagtgcca
601160DNAArtificial SequencePIns 11ccaacatctt tgcggctccc acttggtgga
ggcgctgtac cttgtctgcg gagagcgcgg 601260DNAArtificial SequencePIns
12atcttcggct tcgcgacgag tcttaggggt atagaagaat ccgcgctctc cgcagacaag
601360DNAArtificial SequencePIns 13ctcgtcgcga agccgaagat ctgcaggttg
gtcaggtcga actgggcggc ggccctggtg 601460DNAArtificial SequencePIns
14tgcaaggagc cttccagggc gagtggctgg agggagccgg caccagggcc gccgcccagt
601560DNAArtificial SequencePIns 15gccctggaag gctccttgca aaaacgcgga
atcgtggagc agtgctgtac cagcatctgc 601653DNAArtificial SequencePIns
16tcagttgcag tagttctcaa gttggtagag ggagcagatg ctggtacagc act
531725DNAArtificial Sequenceprimer 17ttcgtgaacc aacatctttg cggct
251834DNAArtificial Sequenceprimer 18gcctctagat cagttgcagt
agttctcaag ttgg 3419261DNAArtificial SequencePIns 19ttcgtgaacc
aacatctttg cggctcccac ttggtggagg cgctgtacct tgtctgcgga 60gagcgcggat
tcttctatac ccctaagact cgtcgcgaag ccgaagatct gcaggttggt
120caggtcgaac tgggcggcgg ccctggtgcc ggctccctcc agccactcgc
cctggaaggc 180tccttgcaaa aacgcggaat cgtggagcag tgctgtacca
gcatctgctc cctctaccaa 240cttgagaact actgcaactg a
2612028DNAArtificial Sequenceprimer 20ggcggtaccc aaattcctgt
gaagtagc 282146DNAArtificial Sequenceprimer 21ccgcaaagat gttggttcac
gaaagcgaat gctgggatag ctacgc 462225DNAArtificial Sequenceprimer
22gatgtcggaa gaaccggttc ccttg 252346DNAArtificial Sequenceprimer
23ccgcaaagat gttggttcac gaactgagcg aatgctggga tagcta
462446DNAArtificial Sequenceprimer 24ccgcaaagat gttggttcac
gaactcctga gcgaatgctg ggatag 462546DNAArtificial Sequenceprimer
25ccgcaaagat gttggttcac gaaggtctcc tgagcgaatg ctggga
462646DNAArtificial Sequenceprimer 26ccgcaaagat gttggttcac
gaagttggtc tcctgagcga atgctg 462746DNAArtificial Sequenceprimer
27ccgcaaagat gttggttcac gaatgggttg gtctcctgag cgaatg
462846DNAArtificial Sequenceprimer 28ccgcaaagat gttggttcac
gaaggttggg ttggtctcct gagcga 462946DNAArtificial Sequenceprimer
29ccgcaaagat gttggttcac gaagaaggtt
gggttggtct cctgag 463046DNAArtificial Sequenceprimer 30ccgcaaagat
gttggttcac gaagttgaag gttgggttgg tctcct 463146DNAArtificial
Sequenceprimer 31ccgcaaagat gttggttcac gaagatgttg aaggttgggt tggtct
463246DNAArtificial Sequenceprimer 32ccgcaaagat gttggttcac
gaagttgatg ttgaaggttg ggttgg 463346DNAArtificial Sequenceprimer
33ccgcaaagat gttggttcac gaagttgttg atgttgaagg ttgggt
463446DNAArtificial Sequenceprimer 34ccgcaaagat gttggttcac
gaagccgttg ttgatgttga aggttg 463546DNAArtificial Sequenceprimer
35ccgcaaagat gttggttcac gaagaagccg ttgttgatgt tgaagg
463646DNAArtificial Sequenceprimer 36ccgcaaagat gttggttcac
gaagttgaag ccgttgttga tgttga 463746DNAArtificial Sequenceprimer
37ccgcaaagat gttggttcac gaaatcgttg aagccgttgt tgatgt
463846DNAArtificial Sequenceprimer 38ccgcaaagat gttggttcac
gaaatcagca tcgttgaagc cgttgt 463946DNAArtificial Sequenceprimer
39ccgcaaagat gttggttcac gaaggtggat ccatcagcat cgttga
464046DNAArtificial Sequenceprimer 40ccgcaaagat gttggttcac
gaagtactgg aactcttcca ggtaag 464146DNAArtificial Sequenceprimer
41ccgcaaagat gttggttcac gaaagtgatg gtctcagcgg aagccg
464246DNAArtificial Sequenceprimer 42ccgcaaagat gttggttcac
gaactctgca aagtcagtct tgttct 464346DNAArtificial Sequenceprimer
43ccgcaaagat gttggttcac gaattcattg ttatcaattg cgaact
464446DNAArtificial Sequenceprimer 44ccgcaaagat gttggttcac
gaagagctgg ggaaccagaa cgtcag 464546DNAArtificial Sequenceprimer
45ccgcaaagat gttggttcac gaagatcagg gaagagtagt gctggc
464646DNAArtificial Sequenceprimer 46ccgcaaagat gttggttcac
gaagatagcg aagtcgagct tagcct 464746DNAArtificial Sequenceprimer
47ccgcaaagat gttggttcac gaagcggagc tgctcagcgc ggtatg
464846DNAArtificial Sequenceprimer 48ccgcaaagat gttggttcac
gaagatgtcg gaagaaccgg ttccct 464934DNAArtificial Sequenceprimer
49gccggtacct cagttgcagt agttctcaag ttgg 345046DNAArtificial
Sequenceprimer 50tggttcacga agcggccctc gatatcagca tcgttgaagc cgttgt
465146DNAArtificial Sequenceprimer 51tggttcacga agcggccctc
gatgtactgg aactcttcca ggtaag 465235DNAArtificial Sequenceprimer
52atcgagggcc gcttcgtgaa ccaacatctt tgcgg 355341DNAArtificial
Sequenceprimer 53gaaaacctgt acttccagtt cgtgaaccaa catctttgcg g
415446DNAArtificial Sequenceprimer 54tggttcacga agcggccctc
gatggttggg ttggtctcct gagcga 465546DNAArtificial Sequenceprimer
55acgaactgga agtacaggtt ttcggttggg ttggtctcct gagcga
465660DNAArtificial SequencehGH 56tttccaacaa tcccgctgag ccgcctcttc
gataacgctt cgctccgcgc tcaccgcctg 605760DNAArtificial SequencehGH
57gtaccaggaa ttcgaggaag cgtatattcc caaggaacag aaatactcgt ttctccaaaa
605860DNAArtificial SequencehGH 58tttccgagtc gattcctacc ccctccaatc
gtgaggaaac ccagcaaaaa agcaacctcg 605960DNAArtificial SequencehGH
59cttcttatcc agtcctggct ggagcccgtg cagtttttgc gcagcgtctt tgctaactct
606060DNAArtificial SequencehGH 60ttccaacgtg tacgatcttt tgaaggatct
cgaagagggt attcagactc tgatgggccg 606160DNAArtificial SequencehGH
61gcacgggcca aattttcaag caaacctaca gcaaatttga tactaactcc cacaatgacg
606260DNAArtificial SequencehGH 62ggtctgctct actgcttctt caaggatatg
gataaggtcg aaaccttcct ccgtatcgtg 606360DNAArtificial SequencehGH
63cttcctcgaa ttcctggtac gtgtcgaacg cgagttggtg caggcggtga gcgcggagcg
606460DNAArtificial SequencehGH 64ggtaggaatc gactcggaaa agcagaggct
ggtttggggg ttttggagaa acgagtattt 606560DNAArtificial SequencehGH
65agccaggact ggataagaag cagtgagata cgcagcaact cgaggttgct tttttgctgg
606660DNAArtificial SequencehGH 66aaagatcgta cacgttggaa tccgacgctc
catacacaag agagttagca aagacgctgc 606760DNAArtificial SequencehGH
67cttgaaaatt tggcccgtgc gaggcgatcc gtcttcgagg cggcccatca gagtctgaat
606860DNAArtificial SequencehGH 68aagaagcagt agagcagacc gtaatttttc
aacaaagcat cgtcattgtg ggagttagta 606956DNAArtificial SequencehGH
69tcagaaaccg cacgagccct ccactgagcg gcactgcacg atacggagga aggttt
567023DNAArtificial Sequenceprimer 70tttccaacaa tcccgctgag ccg
237131DNAArtificial Sequenceprimer 71gccggtacct cagaaaccgc
acgagccctc c 3172585DNAArtificial SequencehGH 72tttccaacaa
tcccgctgag ccgcctcttc gataacgctt cgctccgcgc tcaccgcctg 60caccaactcg
cgttcgacac gtaccaggaa ttcgaggaag cgtatattcc caaggaacag
120aaatactcgt ttctccaaaa cccccaaacc agcctctgct tttccgagtc
gattcctacc 180ccctccaatc gtgaggaaac ccagcaaaaa agcaacctcg
agttgctgcg tatctcactg 240cttcttatcc agtcctggct ggagcccgtg
cagtttttgc gcagcgtctt tgctaactct 300cttgtgtatg gagcgtcgga
ttccaacgtg tacgatcttt tgaaggatct cgaagagggt 360attcagactc
tgatgggccg cctcgaagac ggatcgcctc gcacgggcca aattttcaag
420caaacctaca gcaaatttga tactaactcc cacaatgacg atgctttgtt
gaaaaattac 480ggtctgctct actgcttctt caaggatatg gataaggtcg
aaaccttcct ccgtatcgtg 540cagtgccgct cagtggaggg ctcgtgcggt
ttctgaggta ccggc 5857346DNAArtificial Sequenceprimer 73cggctcagcg
ggattgttgg aaatgccgtt gccacaggtg cggcca 467446DNAArtificial
Sequenceprimer 74cggctcagcg ggattgttgg aaagcgaatg ctgggatagc aacgcc
467546DNAArtificial Sequenceprimer 75cggctcagcg ggattgttgg
aaagcggccc tcgatggttg ggttgg 467646DNAArtificial Sequenceprimer
76cggctcagcg ggattgttgg aaagcggccc tcgatatcag catcgt
467746DNAArtificial Sequenceprimer 77cggctcagcg ggattgttgg
aaagcggccc tcgatgtact ggaact 467860DNAArtificial
SequenceTeriparatide 78agcgtctccg agattcagct tatgcacaac ctgggcaagc
acttgaactc catggagcga 607962DNAArtificial SequenceTeriparatide
79gaagttgtgg acatcttgca gtttctttcg cagccattcg actcgctcca tggagttcaa
60gt 628025DNAArtificial Sequenceprimer 80agcgtctccg agattcagct
tatgc 258133DNAArtificial Sequenceprimer 81gccggtacct cagaagttgt
ggacatcttg cag 3382114DNAArtificial SequenceTeriparatide
82agcgtctccg agattcagct tatgcacaac ctgggcaagc acttgaactc catggagcga
60gtcgaatggc tgcgaaagaa actgcaagat gtccacaact tctgaggtac cggc
1148348DNAArtificial Sequenceprimer 83gcataagctg aatctcggag
acgcttgccg ttgccacagg tgcggcca 488448DNAArtificial Sequenceprimer
84gcataagctg aatctcggag acgctagcga atgctgggat agctacgc
488546DNAArtificial Sequenceprimer 85ccaacccaac catcgagggc
cgcagcgtct ccgagattca gcttat 468625DNAArtificial Sequenceprimer
86gcggccctcg atggttgggt tggtc 258725DNAArtificialprimer
87gtactggaac tcttccaggt aagcg 258846DNAArtificialprimer
88agtacgaaaa cctgtacttc cagagcgtct ccgagattca gcttat
468936DNAArtificialprimer 89gccggtacct catcagaagt tgtggacatc ttgcag
369054DNAArtificialBiva18 90cgcccgggtg gaggtggcaa cggagatttc
gaggagatcc cggaagagta cctg 549151DNAArtificialprimer 91ccgttgccac
ctccacccgg gcgcttgtac tggaactctt ccaggtaagc g
519237DNAArtificialprimer 92gccggtacct catcacaggt actcttccgg
gatctcc 379318PRTArtificialBiva18 93Arg Pro Gly Gly Gly Gly Asn Gly
Asp Phe Glu Glu Ile Pro Glu Glu 1 5 10 15 Tyr Leu
* * * * *
References