Recombinant expression of streptococcus pyogenes cysteine protease and immunogenic compositions thereof
AssigneeWyeth Holdings Corporation
InventorsLaurie Winter, Yury Matsuka, Elzabeth Anderson, Stephen Olmsted
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Abstract
The present invention generally relates to the fields of molecular biology, clinical bacteriology and protein folding. More particularly, the invention relates to methods for recombinantly expressing a soluble mature Streptococcus pyogenesexotoxin B (SpeB) polypeptide in a host cell.
Claims71
A method for recombinantly expressing a mature Streptococcus pyogenes exotoxin B (SpeB) polypeptide in a host cell, the method comprising transforming, transducing, transfecting or infecting a host cell with a polycistronic plasmid, the plasmid comprising (a) a polynucleotide sequence encoding a SpeB pro-polypeptide domain and (b) a polynucleotide sequence encoding a mature SpeB polypeptide, and culturing the host cell under conditions which permit the expression of the mature SpeB polypeptide and the SpeB pro-polypeptide domain by the host cell, and wherein the mature SpeB polypeptide is soluble in the host cell.
A method for recombinantly expressing a mature SpeB polypeptide in a host cell, the method comprising:
- (a) transforming, transducing, transfecting or infecting a host cell with (i) a plasmid comprising a polynucleotide sequence encoding a SpeB pro-polypeptide domain and (ii) a plasmid comprising a polynucleotide sequence encoding a mature SpeB polypeptide;
- and
- (b) culturing the host cell under conditions suitable to co-express the SpeB pro-polypeptide domain and the mature SpeB polypeptide,
- wherein the mature SpeB polypeptide is soluble in the host cell.
A method for producing a mature SpeB polypeptide comprising the steps of:
- (a) recombinantly expressing in a host cell a plasmid comprising a polynucleotide sequence encoding a mature SpeB polypeptide, wherein the SpeB polypeptide forms an insoluble polypeptide aggregate in the host cell;
- (b) solubilizing the polypeptide aggregate, wherein the solubilized polypeptide is defined as a non-native mature SpeB polypeptide;
- (c) refolding the non-native mature SpeB polypeptide in the presence of one or more chaperone proteins, wherein the non-native mature SpeB polypeptide is folded into a native mature SpeB polypeptide;
- and
- (d) recovering the native mature SpeB polypeptide.
A method for recombinantly expressing a mature SpeB polypeptide in a host cell comprising expressing in a host cell a polycistronic plasmid comprising (i) a polynucleotide sequence encoding a mature SpeB polypeptide and (ii) a polynucleotide sequence encoding a GroEL polypeptide, wherein the mature SpeB polypeptide is soluble in the host cell.
A method for producing a mature SpeB polypeptide comprising the steps of:
- (a) transforming, transducing, transfecting or infecting a host cell with a polycistronic plasmid comprising (i) a polynucleotide sequence encoding a mature SpeB polypeptide and (ii) a polynucleotide sequence encoding a GroEL polypeptide;
- (b) culturing the host cell under conditions suitable to express the mature SpeB polypeptide and the GroEL polypeptide, wherein the mature SpeB polypeptide is soluble in the host cell;
- and
- (c) recovering the native mature SpeB polypeptide.
A polycistronic plasmid comprising (a) a polynucleotide sequence encoding a SpeB pro-polypeptide domain and (b) a polynucleotide sequence encoding a mature SpeB polypeptide, wherein the mature SpeB polypeptide is soluble when expressed in a host cell.
A plasmid comprising a polynucleotide sequence encoding a SpeB pro-polypeptide domain and a plasmid comprising a polynucleotide sequence encoding a mature SpeB polypeptide, wherein the mature SpeB polypeptide is soluble when expressed in a host cell.
A polycistronic plasmid comprising (a) a polynucleotide sequence encoding a mature SpeB polypeptide and (b) a polynucleotide sequence encoding a GroEL polypeptide, wherein the mature SpeB polypeptide is soluble when expressed in a host cell.
A polycistronic plasmid comprising (a) a polynucleotide sequence encoding a mature SpeB polypeptide, (b) a polynucleotide sequence encoding a GroEL polypeptide and (c) a polynucleotide sequence encoding a GroES polypeptide, wherein the mature SpeB polypeptide is soluble when expressed in a host cell.
A polycistronic plasmid comprising (a) a polynucleotide sequence encoding a mature SpeB polypeptide and (b) a polynucleotide sequence encoding one or more polypeptides selected from the group consisting of GroEL, GroES, SpeB pro-polypeptide domain, PDI and PPI, wherein the mature SpeB polypeptide is soluble when expressed in a host cell.
Dependencies are inferred from the claim text.