Expression of proteins on bacterial surface
AssigneeGeorge Georgiou
InventorsGeorge Georgiou, Joseph A. Francisco, Charles F. Earhart
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Abstract
The invention relates to a method for producing stable, surface-expressed polypeptides from recombinant gram-negative bacterial cell hosts. A tripartite chimetic gene and its related recombinant vector include separate DNA sequences for directing or targeting and translocating a desired gene product from a cell periplasm to the external cell surface. A wide range of polypeptides may be efficiently surface expressed, including .beta.-lactamase and alkaline phosphatase. Full enzyme activity is maintained and the proteins remain anchored to the bacterial outer membrane surface.
Claims19
A recombinant DNA for expressing a polypeptide stably anchored on the external surface of the outer membrane of E. coli or Salmonella comprising:
- (A) A Salmonella or E. coli lipoprotein 5' gene segment which encodes at least the signal peptide and at least the first three amino acids of the mature protein;
- (B) A DNA segment encoding a transmembrane outer membrane protein selected from the group consisting of OmpA, OmpC, OmpF and OmpT of E. coli or Salmonella;
- and
- (C) A DNA segment encoding a desired soluble heterologous or homologous polypeptide not normally found in the outer membrane of gram negative bacteria wherein DNA (A) is linked 5' to DNA (B) and DNA (B) is linked 5' to DNA (C), all operatively linked with a promoter sequence to express and anchor the desired polypeptide on the external bacterial surface.
A recombinant DNA vector comprising:
- An E. coli or Salmonella lipoprotein 5' gene segment which encodes the signal peptide and at least the first three amino acids of the mature protein;
- A DNA segment encoding a transmembrane outer membrane protein selected from the group consisting of OmpA, OmpT, OmpF and OmpC of E. coli or Salmonella;
- and
- A polylinker DNA segment into which a DNA encoding soluble heterologous or homologous polypeptide not normally found in the outer membrane of gram-negative bacteria may be inserted, all being operatively linked 5' to 3' with a promoter sequence to express and stably anchor the polypeptide to the external bacterial surface.
Dependencies are inferred from the claim text.