Methods for production of proteins in host cells

Details Methods for production of proteins in host cells Methods for production of proteins in host cells

1999-12-23

2004-01-13

Leffers, Jr., Gerald G. (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...

C435S069100, C435S320100, C435S471000, C435S252100, C435S252300, C435S252800, C435S325000, C435S455000, C536S023100, C536S023400

Reexamination Certificate

active

06677139

ABSTRACT:

FIELD OF THE INVENTION
The present invention generally relates to methods for the production of proteins in host cells. Specifically, the present invention relates to the use of a chaperonin binding domain in expression systems designed for the production of proteins in host cells.
BACKGROUND OF THE INVENTION
One of the simplest and most inexpensive ways to obtain large quantities of desired polypeptides for commercial or research uses is through the expression of heterologous genes in bacterial cells. Often however, net accumulation of the recombinant polypeptide is low due to degradation, missfoldings or aggregation. Also, many recombinant polypeptides fail to attain their correct three-dimensional conformation in
E.coli
and are found sequestered within large refractile aggregates, i.e., inclusion bodies. Processes for recovering active polypeptides from inclusion bodies can be complex and expensive. Additionally, in some cases, the large scale production of a protein is limited by the toxicity of the overexpressed protein toward the host cell or the accumulation of proteins as inclusion bodies that impede their recovery and purification.
In the cell, a class of accessory proteins known as molecular chaperones function by interacting with nascent polypeptide chains and aid in the process of correct folding Georgiou et al. (1996, Current Opinion in Biotechnology, 7:190-197). Molecular chaperones are highly conserved proteins found in all organisms that control and sometimes catalyse the ATP-dependent folding of newly synthesized proteins and polypeptides as they are produced in cells. Chaperones mediate the stabilization and refolding of proteins under conditions of stress and are believed to fold crucial portions of proteins, such as enzymes, independently (Hendrick, J. P., 1993
, Ann. Rev. Biochem
. 62: 349-384).
Several
E.coli
proteins have been shown to exhibit chaperone activity: the 60 kDa heat shock protein (Hsp60) GroEL, a chaperonin and the smaller accessory protein GroES (10 kDa); the DnaK (Hsp70), DnaJ and GrpE complex; and the Clp system. Georgiou et al. supra. GroEL consists of 14 subunits which are arranged in two heptameric rings stacked back to back. The central cavity of the cylinder accepts unfolded substrate polypeptides in the conformation of a collapsed intermediate. GroEL interacts with GroES, a single heptameric ring that binds asymetrically to GroEL, capping one opening of the cylinder. GroES coordinates the ATP hydrolysis by GroEL with productive folding (Mayhew, M et al.,
Nature
vol. 379:420-426.)
Dale, G. et al. (1994, Protein Engineering 7:925-931) report that simultaneous overproduction of the GroEL/GroES chaperonins with dihydrofolate reductase results in an increased solubility of the enzyme and Amrein, K. et al. (1995, Proc. Natl. Acad. Sci. vol 92: 1048-1052) report on the purification of recombinant human protein-tyrosine kinase in an
E.coli
expression system overproducing the bacterial chaperonins GroES and GroEL.
Landry, S. et al. (1993, Nature 364:255-258) disclose a polypeptide loop of the GroES/GroEL complex and Altamirano et al. (1997, Proc. Natl. Acad. Sci. USA, 94:3576-3578) disclose the use of immobilized fragments of the GroEL chaperonin in chromatography.
In spite of advances in understanding chaperonins and the production of proteins in host cells, there remains a need to develop expression vectors and systems which allow for production of proteins in host cells.
SUMMARY OF THE INVENTION
The present invention generally relates to chaperonin protein binding domains and to the use of an isolated chaperonin protein binding domain in the production of heterologous proteins, peptides or polypeptides in a host cell. The present invention is based, in part, upon the finding that a toxic gene product could be recombinantly produced by a host cell when expressed as a fusion protein associated with an isolated chaperonin binding domain.
Accordingly, the present invention provides a method for producing a protein in a host cell, comprising the step of culturing a host cell comprising a first nucleic acid encoding an isolated chaperonin binding domain associated with a second nucleic acid encoding the protein and a third nucleic acid encoding a chaperonin under conditions suitable for expression of said first, said second and said third nucleic acid and wherein said chaperonin binding domain is capable of binding to said chaperonin. In a further embodiment, the chaperonin binding domain and the chaperonin are capable of binding with an affinity of between about 10
−2
and 10
−8
Kd. The method may further comprise recovering said protein from said cell. In one aspect, the protein is one toxic to the host cell. A protein may be toxic to a host cell due to its intrinsic nature or toxic due to the presence of elevated levels in the host cell.
In another embodiment of the present invention, the first and second nucleic acid encode a fusion protein. The first and second nucleic acid may be directly linked or indirectly linked by nucleic acid encoding an enzymatic cleavage site, a chemical cleavage site, or another protein or peptide.
In one aspect of the invention, nucleic acid encoding the chaperonin is naturally produced by the host cell and the cell is grown under conditions that result in elevated levels of the chaperonin. In another aspect, nucleic acid encoding the chaperonin is heterologous to the host cell and the heterologous chaperonin is under the control of at least one expression signal capable of overexpressing the chaperonin in the host cell. The present invention encompasses any host cell that is capable of expression of recombinant proteins. In one embodiment, the host cell is a bacterium. In another embodiment, the host cell is a eubacterium. In yet further embodiments, the host cell is a gram-positive or a gram-negative bacterium. In a further embodiment, the bacterial cell is a member of the family Enterobacteriaceae. In an additional embodiment, the bacterial cell is an Escherichia species, in particular
E. coli.
There are several well characterized chaperonin systems known in the art having two or more interacting partners, for example, Hsp60 and Hsp10 (GroEL/GroES); Hsp70 and Hsp40 and GrpE (DnaK/DNAJ/GrpE); ClipA/X and ClipP; Hsp90 and Hsp70 and other factors; TriC and other factors. The present invention encompasses chaperonin binding domains obtainable from these systems as long as the chaperonin binding domain is capable of binding to a chaperonin with an affinity of between about 10
−2
and 10
−8
Kd. In one embodiment, the chaperonin binding domain has the sequence as shown in SEQ ID NO: 3 through SEQ ID NO: 40. In yet another embodiment, the chaperonin binding domain is obtainable from the GroES co-chaperonin and said chaperonin is the GroEL chaperonin. In another embodiment, the binding domain comprises the amino acid sequence EVETKSAGGIVLTGSAAA(SEQ ID NO:2), In a further embodiment, the binding domain comprises a variation of the sequence EVETKSAGGIVLTGSAAA(SEQ ID NO:2), said variant being capable of binding to GroEL chaperonin with an affinity of 10
−2
to 10
−8
Kd. The present invention also provides expression vectors and host cells comprising a chaperonin protein binding domain.
Examples of heterologous proteins include therapeutically significant proteins, such as growth factors, cytokines, ligands, receptors and inhibitors, as well as vaccines and antibodies; enzymes such as hydrolases including proteases, carbohydrases, and lipases; isomerases such as racemases, epimerases, tautomerases, or mutases; transferases, kinases and phophatases; and commercially important industrial proteins or polypeptides, such as proteases, carbohydrases such as amylases and glucoamylases, cellulases, oxidases and lipases. The nucleic acid encoding the heterologous protein may be naturally occurring, a variation of a naturally occurring protein or synthetic.


REFERENCES:
patent: 5302518 (1994-04-01), Neupert et al.
patent: WO 92/05249 (1992-04-01), None
patent: WO 93/13200 (1993-07-

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