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

Abstract

AIM:

AIM:

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.

CONCLUSION:

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.

• Authors: Yun-xia Yang, Yun Feng, Bo-yao Wang, Qi Wu
• Journal: Acta pharmacologica Sinica (Acta Pharmacol Sin) Vol. 25 Issue 2 Pg. 239-45 (Feb 2004) ISSN: 1671-4083 [Print] United States
• PMID: 14769216 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Anti-Bacterial Agents
• Antimicrobial Cationic Peptides
• DNA, Complementary
• FALL 39
• Peptides

Topics

• Amino Acid Sequence
• Anti-Bacterial Agents (pharmacology)
• Antimicrobial Cationic Peptides
• Base Sequence
• DNA, Complementary (genetics)
• Escherichia coli (drug effects, genetics)
• Genetic Vectors
• Hemolysis (drug effects)
• Humans
• Molecular Sequence Data
• Mutagenesis, Site-Directed
• Peptides (genetics, isolation & purification, pharmacology)
• Pseudomonas aeruginosa (drug effects)