Screening Of LPS Binding Site Of C1INH And Expression Of Long-acting Protein C1INH (14-32)-HSA

Sepsis and endotoxin shock, caused by gram-negative bacteria infection, is a clinical problem always. Lipopolysaccharide (LPS) is the main reason in the pathogenesis of sepsis and endotoxin shock. ClINH, a complement regulatory protein, prevents sepsis and endotoxin shock via a direct interaction of the amino-terminal domain with gram-negative bacterial LPS. In this study, we first identified that a fragment within the major part of the amino-terminal domain in in vitro proteolytic analysis of ClINH had an ability to bind to LPS. We synthesized several peptides overlapping the C1INH fragment. Among these synthetic peptides, a13-mer derivative peptide at position from18to30, named N2(18-3o), exhibited the most powerful anti-endotoxin activity in vitro.6amino acids were increased on the two sides of N2(18-30), three of them are hydrophobic amino acids, two low hydrophilic, one hydrophilic. The new peptide was named C1INH (14-32). We developed a long-acting C1INH (14-32)[C1INH (14-32)-HSA] by albumin fusion for prolonging the half-life of ClINH (14-32). The fused gene ClINH (14-32)-HSA was expressed in Pichia pastoris GS115. According to the cDNA sequence of C1INH derived from the GeneBank (NM-182488) and the cDNA sequence of HSA derived from the GeneBank (V00495), two pairs of primers was designed. Using PCR C1INH (14-32) was connected with the HSA gene. Then C1INH (14-32)-HSA was connected with expression vector (pHBM905a) by digestion and connection. The expression vector pHBM-C1INH (14-32)-HSA linearized by Sall restriction endonuclease was transformed into Pichia pastoris GS115by electroporation. And the recombinant strains were screened by HIS limitation and expression level by SDS-PAGE and Western Blotting. These data indicate that the peptide N2(18-30) derived from the amino-terminal region of ClINH is anti-endotoxin. The fusion protein C1INH (14-32)-HAS can been expressed in Pichia pastoris GS115.