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
1999-12-28
2001-03-06
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S252300, C435S252330, C536S023100, C536S024100
Reexamination Certificate
active
06197547
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to an artificial operon, an expression plasmid carrying the operon, a cotransformant harboring an expression vector for a foreign protein and any one of expression plasmids, and a method for producing a foreign protein comprising expressing a foreign protein by the use of the cotransformant, which are capable of expressing a foreign protein in a solubilized form and in a state of having a correct conformation.
2. Discussion of the Related Art
A trigger factor is a protein which has been found as a cytoplasmic factor required for in vitro transporting to a membrane of proOmpA, a precursor of
E. coli
outer membrane protein OmpA [Crooke, E. and Wickner, W.,
Proc. Nat. Acad. Sci. USA
84, 5216-5220 (1987)]. In addition, a tig gene has been cloned as a gene encoding a trigger factor having a molecular weight of 48 kDa [Guthrie, B. and Wickner W.,
J. Bacteriol.
172, 5555-5562 (1990)]. On the basis of analysis of the amino acid sequence, it has been elucidated that the trigger factor has FK506-bound protein (FKBP) domain, and that all of amino acid residues required for each expression of the binding activity with FK506 and for a peptidyl-prolyl isomerase (PPIase) activity are conserved in the trigger factor [Callebaut, I. and Mornon, J. -P.,
FEBS Lett.
374, 211-215 (1995)].
There has been reported that the trigger factor has been also identified as PPIase bound to 50S subunit of
E. coli
ribosome, and that the trigger factor markedly enhances prolyl isomerization in in vitro refolding of mutant RNase T
1
[Stoller, G. et al.,
EMBO J.
14, 4939-4948 (1995)]. Moreover, there has been found by an experiment using a crosslinking reagent that the trigger factor is bound to a nascent polypeptide chain on
E. coli
ribosome [Valent, Q. A. et al.,
EMBO J.
14, 5494-5505 (1995); Hesterkamp, T. et al.,
Proc. Nat. Acad. Sci. USA
93, 4437-4441 (1996)]. In addition, the trigger factor has been known to enhance binding to an unfolded protein of GroEL [Kandror, O. et al.,
EMBO J.
14, 6021-6027 (1995); Kandror, O. et al.,
J. Biol. Chem.
272, 1730-1734 (1997)].
PPIase acts on proline residue in a peptide chain, and catalyzes cis-trans isomerization of conformation regarding a peptide bond. This reaction is considered as a rate-determining step of a folding process of the protein. In addition, it is considered that the PPIase family protein is involved in protein folding, refolding, association and dissociation, transport, and the like, within the cells.
In addition, the trigger factor is shown to assist the folding of several proteins in vitro [Scholz, C. et al.,
EMBO J.
16, 54-58 (1997)]. However, the actual function of the trigger factor has not yet been known.
In expression of a foreign protein by
E. coli
, various efforts have been made on the aggregation suppression and the stabilization of a desired foreign protein by coexpression of chaperones. However, it has been difficult to predict coexpression of which of the chaperones is effective for a particular protein, so that undue experimentation is presently carried out in order to determine the effective chaperone. In addition, there are some cases where sufficient effects cannot be obtained by coexpression of known chaperones.
In view of the above problems, an object of the present invention is to provide an artificial operon comprising genes encoding each of a trigger factor, GroEL and GroES, the artificial operon being capable of expressing a foreign protein in a solubilized form and in a state of having a correct conformation.
In one embodiment, the present invention provides an expression plasmid carrying the operon and an expression plasmid for a trigger factor.
In another embodiment, the present invention provides a cotransformant harboring both of the above expression 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 expressing a foreign protein by the use of the cotransformant.
These and other objects of the present invention will be apparent from the following description.
SUMMARY OF THE INVENTION
In sum, the present invention pertains to the following:
[1] an artificial operon comprising genes encoding each of a trigger factor, GroEL and GroES;
[2] a plasmid capable of expressing each of a trigger factor, GroEL and GroES, the plasmid carrying the artificial operon according to item [1];
[3] a plasmid capable of expressing a trigger factor, the plasmid carrying a gene encoding the trigger factor under control of an inducible promoter;
[4] a cotransformant harboring the plasmid according to item [2] or [3] and an expression plasmid for a foreign protein; and
[5] a method for producing a foreign protein comprising expressing said foreign protein by the cotransformant according to item [4].
REFERENCES:
Hartl, Nature 381: 571-580, Molecular chaperones in cellular protein folding, Jun. 1996.
Wall et al., Current Opinion in Biotechnology 6: 507-516, Effects of overexpressing folding modulators on the in vivo folding of heterologous proteins inE. coli, 1995.
Thomas, Jeffrey G. et al., Applied Biochemistry and Biotechnology, vol. 66 (1997) pp. 198-238.
Yasukawa, Takashi et al., The Journal of Biological Chemistry, vol. 270, No. 43 (1995) pp. 25328-25331.
Crooke et al.,Proc. Natl. Acad. Sci., USA, vol. 84, pp. 5216-5220 (Aug. 1987).
Guthrie et al.,Journal of Bacteriology, vol. 172, No. 10, pp. 5555-5562 (Oct. 1990).
Callebaut et al.,FEBS Letter, vol. 374, pp. 211-215 (1995).
Stoller et al.,The EMBO Journal, vol. 14, No. 20, pp. 4939-4948 (1995).
Valent et al.,The EMBO Journal, vol. 14, No. 22, pp. 5494-5505 (1995).
Hesterkamp et al.,Proc. Natl. Acad. Sci.USA, vol. 93, pp. 4437-4441 (Apr. 1996).
Kandror et al.,The EMBO Journal, vol.14, No. 23, pp. 6021-6027 (1995).
Kandror et al.,The Journal of Biologocial Chemistry, vol. 272, No. 3, pp. 1730-1734 (Jan. 1997).
Scholz et al.,The EMBO Journal, vol. 16, No. 1, pp. 54-58 (1997).
Kohara et al.,Cell, vol. 50, pp. 495-508 (Jul. 31, 1987).
Hemmingsen et al.,Nature, vol. 333, pp. 330-334 (1988).
Nishihara et al.,Appl. Environ. Microbiol., vol. 64, pp. 1694-1699 (1988).
Perez-Perez et al.,Gene, vol. 158, pp. 141-142 (1995).
O'Reilly et al.,Cell, vol. 88, pp. 277-285 (Jan. 24, 1997).
Sogo Kazuyo
Yanagi Hideki
Yura Takashi
Birch & Stewart Kolasch & Birch, LLP
Davis Katharine F
HSP Research Institute, Inc.
Schwartzman Robert A.
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