Mutation and Expression of Human Endotoxin Binding Peptide Gene

Objective To analyze the primary structure of parental human endotoxin binding peptide(EBP),induce mutation at base sites corresponding to the amino acids which might influence the biological activity of EBP,clone the mutant of EBP(mEBP) and study its expression.Methods mEBP gene was predicatively analyzed by DNASIS software,and the mutations(Gln→Lys) at sites 5 and 18 were induced by PCR site-directed mutagenesis.The mEBP gene was cloned into prokaryotic vector pinpoint Xa-3 and transformed to E.coli BL21(DE3) pLysS for fusion expression.The expressed product was identified by Western blot.Results The nucleotides at sites 13 and 52 of mEBP gene were mutated from C to A.The constructed recombinant showed identical sequence to that designed,as proved by digestion with restriction endonuclease and sequencing.The biotinylated fusion protein was expressed in E.coli BL21(DE3) pLysS under induction of IPTG.Western blot showed the binding capacity of expressed product to McAb against biotin.Conclusion The mutant of EBP gene was obtained and successfully expressed in E.coli in a form of fusion protein.