PCR-based site-specific mutagenesis of peptide antibiotics FALL-39 and its biologic activities.
OBJECTIVE



OBJECTIVE
To construct PGEX-1lambdaT-FALL-39 expression vector and its mutant vector, and study the relationship of function and structure.


METHODS
A cDNA encoding mature FALL-39 was cloned from SPCA-1 cell mRNA and the prokaryotic expression vector PGEX-1lambdaT-FALL-39 was constructed. Two kinds of polymerase chain reaction (PCR) for the site-direction mutagenesis were used to construct FALL-39 mutant expression vector, FALL-39-Lys-32 and FALL-39-Lys-24. Minimal effective concentration, minimal inhibitory concentration, and minimal bactericidal concentration were used to assay the antibacterial activities of these peptides. Effects of different solution on the antibacterial activity of FALL-39 and FALL-39-Lys-32 were observed by CFU determination. The hemolytic effects of these peptides were also examined on human red blood cells.


RESULTS
Two site-specific mutants FALL-39-Lys-32 and FALL-39-Lys24 were obtained by PCR-induced mutagenesis. In comparison with two-step PCR which required two pairs of primers, one step PCR which required one pair of primers is a simple and efficient method for the PCR based site-specific mutagenesis. Using the prokaryotic expression system, the E coli-based products of recombinant FALL39 and its mutant peptides were also obtained. The antibacterial assay showed that FALL-39-Lys-32 and FALL-39-Lys24 were more potential in the antibacterial activity against E coli ML35p and Pseudomonas aeruginosa ATCC27853 than that of FALL-39, and no increase in hemolysis was observed at the antibacterial concentrations. The antibacterial activity of FALL-39-Lys-32 against E coli was more potent than that of FALL-39 in NaCl-containing LB medium, while its activity was almost the same as FALL-39 in SO4(2-) containing Medium E.


CONCLUSIONS
PCR-based mutagenesis 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 of FALL-39 without increasing hemolysis at the antibacterial concentrations.
Yang
Yun-xia
YX
Department of Pharmacology, West-China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Feng
Yun
Y
Wang
Bo-yao
BY
Wu
Qi
Q
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D005822:Genetic Vectors; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D011550:Pseudomonas aeruginosa / Q000187:drug effects
D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D018076:DNA, Complementary; C091425:FALL 39; D010455:Peptides