Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-04-08
2003-12-16
Devi, S. (Department: 1645)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S023700, C435S069700, C435S071100, C435S069300, C435S069800, C435S320100
Reexamination Certificate
active
06664386
ABSTRACT:
FIELD OF THE INVENTION
The subject invention is directed generally to a system for efficient secretion of recombinant proteins, and more particularly to a system which uses an engineered bacterium having a type III secretion system to export properly folded recombinant proteins into a culture supernatant.
BACKGROUND OF THE INVENTION
Throughout this application various publications are referenced, many in parenthesis. Full citations for each of these publications are provided at the end of the Detailed Description. The disclosures of each of these publications in their entireties are hereby incorporated by reference in this application.
Advances in recombinant DNA biotechnology have made it possible to produce a wide variety of useful polypeptide/protein products in host cells which have been transformed and transfected with DNA sequences which code for production of the polypeptide/protein products. Thus hormones (such as insulin, and growth hormones such as human growth hormone), and industrial or therapeutically useful enzymes (such as chymosin and tissue plasminogen activator (tPA)) have been produced using recombinant DNA technology.
Bacterial cells, in particular
Escherichia coli,
have been used as host cells for the production of recombinant polypeptide/protein products. The genetic systems of such bacterial cells are relatively well understood and such cells exhibit good growth characteristics. However, when such bacterial cells are used to overproduce foreign proteins, the foreign products typically accumulate within the host cells and it is usually necessary to disrupt the cells to effect recovery of the products. Also, recombinant products are often produced within bacterial host cells in the form of insoluble aggregates in which the polypeptides are not in their native, biologically functional form. It is necessary, therefore, to solubilize and denature/renature the insoluble polypeptide products to obtain useful products in soluble, native, biologically functional form. The processes of cell disruption and denaturation/renaturation add significantly to the cost of producing recombinant polypeptide products.
Attempts have been made, therefore, to develop bacterial expression systems which secrete recombinant products into the extracellular culture medium. For example, recombinant heterologous polypeptides have been expressed in bacteria as fusion proteins in which the heterologous polypeptide sequence is joined with an N-terminal signal sequence. However, such fusion proteins, although exported across the inner membrane in Gram-negative bacteria with concomitant removal of the signal sequence, fail to cross the outer membrane and therefore remain within the periplasm. Thus, it is still necessary to disrupt the host cells to effect recovery of heterologous recombinant products and denaturation/renaturation treatment may be required to yield products in native, biologically functional form.
‘Leaky’ mutants of Gram-negative bacterial host cells such as
Escherichia coli
have been proposed for use in the production and secretion of products to the extracellular medium. However, such mutant cells are often not suitable for large scale production of heterologous protein products since the yield of product is generally low and the fragility of the cells makes them unsuitable for growing on a large scale.
U.S. Pat. No. 5,143,830 attempts to address these problems by providing a process for the production of a polypeptide in which host cells are transformed with DNA coding for a fusion protein comprising the polypeptide and a further peptide comprising a C-terminal secretion sequence. The host cells are cultured to express and secrete the fusion protein therefrom. The C-terminal secretion sequence denotes a sequence of amino acids present in the C-terminus of a secreted polypeptide which sequence comprises essential information required for recognition and secretion of the secreted polypeptide via its secretion pathway. Preferably, the C-terminal secretion sequence is a haemolysin C-terminal secretion sequence (haemolysin is an extracellular protein toxin which is produced by some strains of
Escherichia coli
).
U.S. Pat. No. 5,159,062 also attempts to address these problems by providing a signal peptide from
Bordetella pertussis
which can be used for secretion of peptides in
Escherichia coli.
Salmonella spp. have a specialized protein secretion system encoded at centisome 63 of the bacterial chromosome (reviewed in Galan 1996). This protein secretion system, termed type III, directs the export of a number of proteins. Characteristic features of this protein secretion system, which has also been identified in several other animal and plant pathogenic bacteria, include: 1) the absence in the secreted proteins of a typical, cleavable, sec-dependent, signal sequence; 2) the requirement of several accessory proteins for the export process; 3) the export of the target proteins through both the inner and outer membranes; and 4) the requirement of activating extracellular signals for its full function (reviewed in Galan 1996).
Currently known targets of bacterial type III secretion systems include the SptP, SipA, SipB, SipC, SipD, InvJ, SpaO, AvrA, and SopE proteins of Salmonella, the YopE, YopH, YopM and YpkA proteins of Yersinia spp. (Rosqvist et al. 1994; Sory and Cornelis 1994; Persson et al. 1995; Sory et al. 1995; Hakansson et al. 1996), the Ipa proteins of Shigella, and the ExoS proteins of
Pseudomonas aeruginosa.
Given the continuing need and desire to efficiently produce large amounts of recombinant proteins, any system that can produce such large amounts of biologically functional proteins remains desirable.
SUMMARY OF THE INVENTION
The subject invention addresses this need by providing a signal peptide comprising an amino acid sequence at least 90% homologous to the amino acid sequence as shown in SEQ ID NO:1. In one embodiment, the amino acid sequence of the signal peptide is as shown in SEQ ID NO:1. Further provided is a fusion protein comprising the signal peptide fused to a heterologous protein. Also provided are nucleic acid molecules encoding the signal peptide, and encoding the fusion protein, as well as vectors and recombinant host cells comprising the nucleic acid molecules. The recombinant host cell can be a recombinant bacterium having a functional type III secretion system and having loss-of-function mutations in genes that encode secreted substrate proteins of the type III secretion system.
The recombinant host cell can be used in a method of producing a heterologous protein which method comprises culturing the recombinant host cell in a culture medium so as to obtain expression and secretion of the heterologous protein into the culture medium. The secreted heterologous protein can then be recovered from the culture medium.
Also provided is an isolated nucleic acid molecule of at least 45 nucleotides which specifically hybridizes with an isolated nucleic acid molecule having SEQ ID NO:2.
The recombinant bacterium useful for producing heterologous proteins in accordance with the subject invention is also provided. The recombinant bacterium preferably has a functional type III secretion system and has loss-of-function mutations in genes that encode secreted substrate proteins of the type III secretion system.
DETAILED DESCRIPTION OF THE INVENTION
The term “nucleic acid”, as used herein, refers to either DNA or RNA. “Nucleic acid sequence” or “polynucleotide sequence” refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′ end. It includes both self-replicating plasmids, infectious polymers of DNA or RNA, and nonfunctional DNA or RNA.
“Isolated” nucleic acid refers to nucleic acid which has been separated from an organism in a substantially purified form (i.e. substantially free of other substances originating from that organism), and to synthetic nucleic acid.
By a nucleic acid sequence “homologous to” or “complementary to”, it is meant a nucleic acid that selectively hybrid
Galan Jorge E.
Hardt Wolf-Dietrich
Devi S.
Rogalsky & Weyand, LLP
The Research Foundation of State University of New York
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