E.coli-Based Production Of Reconbinant FALL-39 And PCR-based Site-specific Mutagensis And Their Biologic Activities

OBJECTIVE: To construct FALL-39 and its mutant prokaryotic expression vectors, and to study the relationship of function and structure. METHODS: A cDNA encoding mature FALL-39 was cloned from SPCA-1 cell mRNA by using RT-PCR and the E.coli-based prokaryotic expression vector pGEX-lXT-FALL-39 was constructed. Two kinds of polymerase chain reaction (PCR) for the site-direction mutagenesis were used to construct pGEX-lA,T-FALL-39 mutant expression vector, pGEX-UT-FALL-39-Lys32, pGEX-RT-FALL-39-Lys24 andpGEX-lAT-FALL-39-Lys24'32. Using affinity chromatography, thrombin cleaving, AU-PAGE elution, and HPLC, the purified peptides of the native recombinant FALL-39 and its mutants peptides, FALL-39-Lys32, FALL-39-Lys24 and FALL-39-Lys24'32, were obtained. MEC, MIC and MBC were used to assay the antibacterial activities of these peptides. Effects of different solution on the antibacterial activity of FALL-39 and FALL-39-Lys32 were observed by CPU determination. The haemolytic effects of these peptides were also examined on human red blood cells. RESULTS: pGEX-RT-FALL-39 was successfully constructed by DNA sequences analysis. Three site-specific mutants were obtained byPCR-induced mutagenesis. In comparison with two-step PCR required two pairs of primers, one step PCR required one pair of primers is a simple and efficient method for the PCR-based site-specific mutagensis. Using the prokaryotic expression system, the E.coli-based products of recombinant FALL39 and its mutant peptides, FALL-39-Lys32, FALL-39-Lys24 and FALL-39-Lys24'32, were obtained. The antibacterial assay showed that mutant peptides were more potent in the antibacterial activity against E. coll ATCC 25922, E.coli ML 35p and Pseudomonas aeruginosa ATCC 27853 than that of FALL-39, and no increase in haemolysis were observed at the antibacterial concentrations. The antibacterial activity of FALL-39-Lys32 against E.coli ML35p was more potent than that of FALL-39 in NaCl-containing LB medium, while its activity was almost the same as FALL-39 in SO42~ containing Medium E. Inhibitory effect of FALL-39-Lys32 was observed on LPS-induced iNOS mRNA exprssion. CONCLUSION: PCR-based mutagensis is a useful model system for studying the structure and function relationship of antimicrobial peptides. Keeping a -helical conformation of FALL-39 and increasing net positive charge can increase the antibacterial activity and antiendotoxic effect of FALL-39 without increasing haemolysis at the antibacterial concentrations.