Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-09-09
2004-01-06
Crouch, Deborah (Department: 1632)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069700, C536S023500, C536S023600, C536S023700
Reexamination Certificate
active
06673569
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a DsbA/DsbB/DsbC/DsbD expression plasmid. More particularly, the present invention relates to an artificial operon comprising polynucleotides encoding each of DsbA, DsbB, DsbC and DsbD, the operon being capable of expressing a foreign protein in a soluble form while maintaining a normal conformation, an expression plasmid carrying the operon, a cotransformant harboring the expression plasmid and an expression vector for a foreign protein as well as a method for producing a foreign protein comprising culturing the cotransformant.
2. Discussion of the Related Art
Many of the eucaryote-derived proteins have disulfide bonds, and they are not usually expected to have a natural tertiary structure when expressed in the cytoplasm of
E. coli
under strong reductive conditions. Therefore, in the production of such a protein, it is considered to be effective to perform secretory expression into the periplasm under oxidative conditions suitable for disulfide bond formation. In addition to a strong possibility of expressing a protein having its natural conformation, there can be expected various advantages by expression via secretion, including a possibility of expressing a protein which is toxic to cells; a possibility of expressing a protein in which methionine is not added at its N-terminal; and facilitation in purification owing to a reduced amount of contaminant proteins. However, various reports on attempts on secretion of heterologous proteins into the periplasm of
E. coli
have been made, but not all heterologous proteins can be expressed in the forms exhibiting their activities. This is especially a problem in a case of a protein having a large number of disulfide bonds.
On the other hand, in
E. coli
, there have been deduced the roles of DsbA, DsbB, DsbC and DsbD which are the Dsb family proteins involved in the formation of disulfide bonds by means of biochemical tests and complementary tests using their respective deletion strains [Bardwell, J. C.,
Mol. Microbiol.
14, 199-205 (1994); Sone, M. et al.,
J. Biol. Chem.,
272, 10349-10352 (1997); Rietsch, A. et al.,
Proc. Natl. Acad. Sci. USA,
93, 13048-13053 (1996)].
First, DsbA acts to form disulfide bonds in a nascent polypeptide chain which has been transferred into the periplasm. The disulfide bonds formed at this stage are not necessarily proper, and are then corrected into proper disulfide bonds by means of cleavage of the disulfide bonds followed by re-crosslinking by the action of DsbC. Each of DsbA and DsbC has a thioredoxin-like active site motif (Cys-X-X-Cys). In the Cys-X-X-Cys motif, 2 Cys residues are considered to participate in the reaction. In the process of the disulfide bond formation, the 2 Cys residues in the active center of DsbA oxidize a substrate peptide chain, while they themselves are reduced. Two Cys residues in the active center of DsbC are cleaved as a result of the reduction of the disulfide bonds of the substrate once formed, while they themselves are oxidized. Since a reduced form of DsbA and an oxidized form of DsbC no longer have catalytic activities, a factor for re-activating these DsbA and DsbC is necessitated. Intracellular membrane protein DsbB re-oxidizes DsbA, and intracellular membrane protein DsbD re-reduces DsbC, respectively, by action of the thioredoxin-like motifs existing in the periplasmic side.
For the purpose of improving secretion of a desired protein into the periplasm, several attempts have been made to overexpress DsbA or DsbC together with a desired protein, which could not so far be said to be successful. For example, Knappik et al. disclose that DsbA is required for the folding of an expressed product in the secretion of an antibody fragment; however, there has yet remained to be a problem that the efficiency of the folding does not change even when overexpressed [Knappik, A. et al.,
Bio/Technol.,
11, 77-83 (1993)]. In addition, Wunderlich and Glockschuber disclose that the folding of an &agr;-amylase/trypsin inhibitor is not improved by the overexpression of DsbA, but increased to 14 times in the presence of a reductive form of glutathione [Wunderlich, M. and Glockschuber, R.,
J. Biol. Chem.,
268, 24547-24550 (1993)]. Further, Wulfing and Pluckthum disclose that the overexpression of DsbA exhibits some effects on the expression in soluble form of a T cell receptor fragment in the periplasm; however, it is necessary to overexpress simultaneously a heat shock sigma factor &sgr;
32
in addition to DsbA [Wulfing, C. and Pluckthum, A.,
J. Mol. Biol.,
242, 655-669 (1994)]. More recently, Joly et al. have found that the overexpression of DsbA or DsbC serves to doubly increase the expression level of an insulin-like growth factor I (IGF-I) in the periplasm; however, there remains the disadvantage that a soluble expression product is reduced contrary to expectations [Joly, J. C. et al.,
Proc. Natl. Acad. Sci. USA,
95, 2773-2777 (1998)].
An object of the present invention is to provide an artificial operon comprising polynucleotides encoding each of DsbA, DsbB, DsbC and DsbD, the operon being capable of expressing a foreign protein in a soluble form while maintaining a normal tertiary structure.
In one embodiment, the present invention provides an expression plasmid carrying the operon.
In another embodiment, the present invention provides a cotransformant harboring the plasmid and an expression vector for a foreign protein.
In still another embodiment, the present invention provides a method for producing a foreign protein comprising culturing the cotransformant.
These and other objects of the present invention will be apparent from the following description.
SUMMARY OF THE INVENTION
One of the subject matter of the present invention is in the findings that an accurate disulfide bond formation in the periplasm can be surprisingly efficiently carried out, and a soluble expression product can be further efficiently obtained when an expression vector of the Dsb family proteins comprising a protein (DsbA or DsbC) for forming or isomerizing disulfide bonds, as well as a protein (DsbB or DsbD) which can control the reactivity of DsbA or DsbC is constructed and the coexpression effects of these proteins in the secretion of a foreign protein are studied.
In sum, the present invention pertains to the following:
[1] an artificial operon comprising polynucleotides encoding each of DsbA, DsbB, DsbC and DsbD;
[2] an expression plasmid carrying the artificial operon according to item [1] above, usable for expression of DsbA, DsbB, DsbC and DsbD;
[3] a cotransformant harboring the expression plasmid according to item [1] above and an expression vector for a foreign protein; and
[4] a method for producing a foreign protein comprising culturing the cotransformant according to item [3] above.
REFERENCES:
patent: 6159708 (2000-12-01), Sogo et al.
patent: 6197547 (2001-03-01), Sogo et al.
patent: WO 9614422 (1996-05-01), None
A Rietsch et al., Proc.Natl.Acad.Sci USA, “An in vivo pathway for disulfide bond isomerization inEscherichia coli,” Nov. 1996, vol. 93, pp. 13048-13053.*
R Metheringham et al., Mol.Gen Genet, “Effects of mutations in genes for proteins involved in disulphide bond formation in the periplasm on the activities of anaerobically induced electron transfer chains inEscherichia coliK12,” 1996, 253:95-102.*
D Missiakas et al., “Identification and characterization of a new disulfide isomerase-like protein (DsbD) inEscherichia coli,” pp. 3415-3424.*
Dominique Missiakas et al., EMBO Journal, vol. 14, No. 14, pp. 3415-3424 (1995).
Satoshi Kishigami et al., Genes to Cells, vol. 1, pp. 201-208 (1996).
R. Metheringham et al., Molecular and General Genetics, vol. 253, No. 1-2, pp. 95-102 (1996).
J.C. Bardwell, Mol. Microbiol. 14(2),pp. 199-205 (1994).
M. Sone et al., J. Biol. Chem., 272(16),pp. 10349-10352 (1997).
A. Rietsch et al., Proc. Natl. Acad. Sci. USA, 93, pp. 13048-
Kurokawa Yoichi
Yanagi Hideki
Yura Takashi
Crouch Deborah
HSP Research Institute, Inc.
Woitach Joseph
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