Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for...
Reexamination Certificate
1999-11-16
2001-10-23
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
C435S201000, C435S209000, C435S208000, C435S195000, C435S198000, C435S193000, C435S069100, C435S233000, C435S252300, C435S252310, C435S320100
Reexamination Certificate
active
06306631
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a novel ppGpp synthetase and expression systems for improved production of proteins of interest in Gram-positive bacteria, involving use of said novel ppGpp synthetase.
DESCRIPTION OF THE RELATED ART
In gram-positive bacteria secreted proteins are exported across a cell membrane and a cell wall, and are then subsequently released into the external medium.
Gram-positive bacteria such as
B. subtilis, B. amyloliquefaciens, B. licheniformis
have a high capacity for secreting proteins, and indeed, many bacillar extracellular enzymes are used industrially.
A number of proteins are involved in the secretion machinery in gram-positive cells.
WO 94/19471 describes a PrsA protein involved in the secretion machinery of Bacillus, and further describe an expression system for enhancing secretion of exoproteins of interest involving over-expression of the PrsA protein.
Dedhia et al. (Biotechnology and Bioengineering 53:379-386 (1997)) describe gram-negative
E. coli
strains which express smaller than wild-type amounts of ppGpp synthetase (relA). Such gram-negative
E. coli
strains give improved production of a chloramphenicol acetyltransferase (CAT) protein.
SUMMARY OF THE INVENTION
The problem to be solved, by the present invention is to provide an expression system for improved production of a protein of interest in a Gram-positive bacterium, e.g. in a Bacillus strain.
The solution is based on cloned novel DNA sequences from Bacillus species
B. subtilis
(SEQ ID No 1) and
B. amyloliquefaciens
(SEQ ID No 3) which both encode a polypeptide having ppGpp synthetase activity.
The novel DNA sequence information disclosed herein can be used to clone similar DNA sequences from Gram positive bacteria by e.g. standard PCR technology. See e.g. a working example herein (vide infra).
This DNA sequence information provides the possibility of constructing an expression system for improved production of at least one protein of interest in a gram-positive bacterium, wherein said gram-positive bacterium is expressing smaller than wild-type amounts of a polypeptide having ppGpp synthetase activity.
Accordingly, in a first aspect, the present invention provides an isolated polynucleotide molecule selected from the group consisting of (a) polynucleotide molecules encoding a polypeptide having ppGpp synthetase activity and comprising a sequence of nucleotides as shown in SEQ ID NO: 1 from nucleotide 21 to nucleotide 2225; (b) species homologs of (a); (c) polynucleotide molecules that encode a polypeptide having ppGpp synthetase activity that is at least 70% identical to the amino acid sequence of SEQ ID NO: 2 from amino acid residue 1 to amino acid residue 734; (d) molecules complementary to (a), (b) or (c); and (e) degenerate nucleotide sequences of (a), (b), (c) or (d).
Within a second aspect of the invention there is provided an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment selected from the group consisting of (a) polynucleotide molecules encoding a polypeptide having ppGpp synthetase activity and comprising a sequence of nucleotides as shown in SEQ ID NO: 1 from nucleotide 21 to nucleotide 2225; (b) species homologs of (a); (c) polynuicleotide molecules that encode a polypeptide having ppGpp synthetase activity that is at least 70% identical to the amino acid sequence of SEQ ID NO: 2 from amino acid residue 1 to amino acid residue 734; and (d) degenerate nucleotide sequences of (a), (b), or (c); and a transcription terminator.
Within a third aspect of the present invention there is provided a cultured cell into which has been introduced an expression vector as disclosed above, wherein said cell expresses the polypeptide encoded by the DNA segment.
A fourth aspect of the present invention provides an isolated polypeptide having ppGpp synthetase activity selected from the group consisting of (a) polypeptide molecules comprising a sequence of amino acid residues as shown in SEQ ID NO:2 from amino acid residue 1 to amino acid residue 734; (b) species homologs of (a).
Within another aspect of the present invention there is provided a composition comprising a purified polypeptide according to the invention in combination with other polypeptides.
Within yet another aspect of the present invention there are provided methods for producing a polypeptide according to the invention comprising culturing a cell into which has been introduced an expression vector as disclosed above, whereby said cell expresses a polypeptide encoded by the DNA segment and recovering the polypeptide.
In a further aspect, the present invention relates to an expression system for improved production of at least one protein of interest in gram-positive bacteria, which expression system comprises a gram-positive bacterium expressing smaller than wild-type amounts of a polypeptide according to the invention.
In a final aspect the present invention relates to a method for improved production of at least one protein of interest in a gram-positive bacterium, which method comprises:
i) culturing an expression system according to the invention; and
ii) purifying said protein of interest from the resulting culture broth or expression system.
DEFINITIONS
Prior to discussing this invention in further detail, the following terms will first be defined
The term “species homolog” is intended to include “ortholog” and/or “paralog”.
The term “ortholog” denotes a polypeptide or protein obtained from one species that has homology to an analogous polypeptide or protein from a different species.
The term “paralog” denotes a polypeptide or protein obtained from a given species that has homology to a distinct polypeptide or protein from that same species.
The term “expression vector” denotes a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. The expression vector of the invention may be any expression vector that is conveniently subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which the vector it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
The term “recombinant expression” or “recombinantly expressed” used herein in connection with expression of a polypeptide or protein is defined according to the standard definition in the art. Recombinant expression of a protein is generally performed by using an expression vector as described immediately above. The term “isolated”when applied to a polynucleotide molecule, denotes that the polynucleotide has been removed from its natural genetic environment and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5′ and 3′ untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan,
Nature
316:774-78, 1985). The term “an isolated polynucleotide” may
Andersen Jens Tonne
Ehrlich Stanislas Dusko
Lambiris Esq. Elias J.
Monshipouri M.
Novozymes A/S
Prouty Rebecca E.
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