FUSION PROTEIN CONTAINING A SINGLE-STRANDED DNA BINDING PROTEIN AND METHODS FOR EXPRESSION AND PURIFICATION OF THE SAME

摘要

The present invention provides an expression vector comprising a promoter and a polynucleotide sequence encoding a fusion protein. The present invention further provides a method for purification of an interest protein. The present invention also provides a fusion protein comprising a single-stranded DNA binding protein and an interest protein or polypeptide fused directly or indirectly with the COOH-terminus or NH2-terminus of the single-stranded DNA binding protein, wherein said fusion protein is capable of binding to single-stranded DNA.

主权项

1. A method for purification of an interest protein, said method comprising:
contacting a host cell with an expression vector, wherein the expression vector comprises:
a promoter and a polynucleotide sequence encoding a fusion protein; wherein the fusion protein-encoding polynucleotide sequence is so operably linked to the promoter that the transcription of the fusion protein-encoding polynucleotide sequence is controlled by the promoter; wherein the fusion protein comprises a single-stranded DNA-binding protein and an interest protein or polypeptide fused directly or indirectly with the COOH-terminus or NH2-terminus of the single-stranded DNA-binding protein;wherein the single-stranded DNA-binding protein is capable of binding to single-stranded DNA; andwherein the interest protein or polypeptide does not contain a physical feature that can be used for affinity purification; culturing the host cell under such conditions that the fusion protein is expressed; lysing the host cell to obtain a cell lysate; contacting the cell lysate with a substrate immobilized with single-stranded DNA to allow the fusion protein to bind to the single-stranded DNA of the substrate; washing the substrate to remove impurities; and eluting the bound fusion protein from the substrate; so that the interest protein or polypeptide can be purified as a part of the fusion protein.