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
2000-03-22
2003-02-18
Murthy, Ponnathapuachuta (Department: 1652)
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, C435S252310, C435S189000
Reexamination Certificate
active
06521421
ABSTRACT:
FIELD OF THE INVENTION
The present invention generally relates to expression of proteins in gram-positive microorganisms and specifically to gram positive microorganism disulfide bond isomerases. The present invention provides expression vectors, methods and systems for the production of proteins in gram-positive microorganisms.
BACKGROUND OF THE INVENTION
Gram-positive microorganisms, such as members of the group Bacillus, have been used for large-scale industrial fermentation due, in part, to their ability to secrete their fermentation products into the culture media. In gram-positive bacteria, secreted proteins are exported across a cell membrane and a cell wall, and then are subsequently released into the external media usually obtaining their native conformation.
The folding of polypeptide chains depends upon the assistance of molecular chaperones and folding catalysts, such as disulfide bond isomerases, which accelerate specific steps in folding. (Hartl et al., 1995, Current Opinion in Structural Biology, 5:92-102). Disulfide bond isomerases can covalently modify proteins by catalyzing specific isomerization steps that may limit the folding rate of some proteins (PCT/US93/09426).
Disulfide bond isomerase catalyzes thiol/disulfide interchange reactions and promotes disulfide formation, isomerization or reduction, thereby facilitating the formation of the correct disulfide pairings, and may have a more general role in the prevention and premature misfolding of newly translocated chains. Disulfide bond isomerase interacts directly with newly synthesized secretory proteins and is required for the folding of nascent polypeptides in the endoplasmic reticulum (ER) of eukaryotic cells.
In spite of advances in understanding portions of the protein secretion machinery in procaryotic cells, the complete mechanism of protein secretion and the mechanisms associated with correct folding of secreted proteins, especially for gram-positive microorganisms has yet to be fully elucidated.
SUMMARY OF THE INVENTION
The capacity of the secretion machinery of a Gram-positive microorganism may become a limiting factor or bottleneck to the secretion of properly folded proteins or polypeptides and the production of proteins having the correct conformation in secreted form, in particular when the proteins are recombinantly introduced and overexpressed by the host cell. The present invention provides a means for alleviating that bottle neck.
The present invention is based, in part, upon the discovery of a
Bacillus subtilis
disulfide bond isomerases, Dsb1 and Dsb2, identified in heretofore uncharacterised translated genomic DNA by their overall amino acid homology with an
E. coli
disulfide bond isomerase. For
E. coli
disulfide bond isomerase having accession number P30018 and ID number DSBB
—
ECOLI
, it was noted that cysteine residues at 41, 44, 104 and 130 were essential for activity. Those residues are conserved in
B. subtilis
Dsb1 and Dsb2 and noted in
FIGS. 2
,
3
and
4
. The present invention is also based upon the overall structural homology that Dsb1 and Dsb2 have with each other. The present invention is also based in part on the presence in of potential transmembrane spanning regions in Dsb1 and Dsb2.
The present invention provides isolated nucleic acid and amino acid sequences for
B. subtilis
Dsb1 and Dsb2. The amino acid sequence for
B. subtilis
Dsb1 and Dsb2 are shown in
FIGS. 1 and 7
, respectfully. The nucleic acid sequences encoding
B. subtilis
Dsb1 and Dsb2 are the polynucleotide sequences shown in
FIGS. 1 and 7
, respectfully.
The present invention also provides improved methods for secreting correctly folded proteins from gram-positive microorganisms. Accordingly, the present invention provides an improved method for secreting a protein in a gram-positive microorganism comprising the steps of obtaining a gram-positive microorganism host cell comprising nucleic acid encoding Dsb1 and/or Dsb2 wherein said nucleic acid is under the control of expression signals capable of expressing said disulfide bond isomerase in a gram-positive microorganism said microorganism further comprising nucleic acid encoding said protein; and culturing said microorganism under conditions suitable for expression of said disulfide bond isomerase and expression and secretion of said protein. In one embodiment of the present invention, the protein is homologous or naturally occurring in the gram-positive microorganism. In another embodiment of the present invention, the protein is heterologous to the gram-positive microorganism.
The present invention provides expression vectors and host cells comprising isolated nucleic acid encoding a gram-positive Dsb1 and/or Dsb2. In one embodiment of the present invention, the host cell comprising nucleic acid encoding Dsb1 or Dsb2 is genetically engineered to produce a desired protein, such as an enzyme, growth factor or hormone. In yet another embodiment of the present invention, the enzyme is selected from the group consisting of proteases, carbohydrases including amylases, cellulases, xylanases, reductases and lipases; isomerases such as racemases, epimerases, tautomerases, or mutases; transferases, kinases and phophatases acylases, amidases, esterases, oxidases. In a further embodiment the expression of the disulfide bond isomerases Dsb1 and/or Dsb2 is coordinated with the expression of other components of the secretion machinery. Preferably other components of the secretion machinery, i.e., translocase, SecA, SecY, SecE and/or other secretion factors known to those of skill in the art are modulated in expression at an optimal ratio to Dsb1 and Dsb2. For example, it may be desired to overexpress multiple secretion factors in addition to Dsb1 or Dsb2 for optimum enhancement of the secretion patterns.
The present invention also provides a method of identifying homologous non
Bacillus subtilis
Dsb1 and Dsb2 that comprises hybridizing part or all of dsb1 and dsb2 nucleic acid shown in the Figures with nucleic acid derived from gram-positive microorganisms. In one embodiment, the nucleic acid is of genomic origin. In another embodiment, the nucleic acid is a cDNA. The present invention encompasses novel gram-positive microorganism disulfide bond isomerases identified by this method.
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Contente et al., “Maker Rescue Tran
Caldwell Robert M.
Quax Wilhelmus J.
Genencor International Inc.
Genencor International, Inc
Kerr Kathleen
Murthy Ponnathapuachuta
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