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Expression and characterization of antimicrobial peptide CecropinAD in the methylotrophic yeast Pichia pastoris

Paper ID Volume ID Publish Year Pages File Format Full-Text
35644 45100 2009 6 PDF Available
Title
Expression and characterization of antimicrobial peptide CecropinAD in the methylotrophic yeast Pichia pastoris
Abstract

Hybrid antibacterial peptide CecropinAD (CAD) is a linear cationic peptide that has potent antimicrobial properties without hemolytic activity. To explore a new approach to express the hybrid peptide CAD in the methylotrophic yeast Pichia pastoris, the cDNA sequence encoding CAD was obtained by recursive PCR (rPCR) and cloned into the vector pPICZα-A. The Sac I-linearized recombinant plasmid pPICZα-CAD was transformed into P. pastoris GS115 by electroporation. Expression of recombinant CAD was induced for 96 h with 1.0% methanol at 28 °C, pH 5.0. The recombinant CAD was purified by two steps of reversed-phase HPLC and 1.8 mg pure active CAD was obtained from 100 ml culture. Tricine-SDS-PAGE and mass spectrometry analyses demonstrated that the molecular weight of the purified CAD was 3.8 kDa. Analysis of circular dichroism (CD) revealed that CAD mainly has α-helixes in the presence of 10 mM phosphate buffer (pH 7.2), 50% TFE/water solution (pH 2.0), or 30 mM SDS (pH 10.8). FACScan analysis showed that the antibacterial mechanism of CAD is to act on the cell membrane to disrupt bacterial cell structure. Antimicrobial assays demonstrated that recombinant CAD has a broad spectrum of anti-microbial property against fungi, as well as Gram-positive and Gram-negative bacteria, but does not have hemolytic activity against human erythrocytes. Our results suggest that recombinant antimicrobial peptide CAD may serve as an attractive candidate for the development of therapeutic antimicrobial drugs.

Keywords
CecropinAD (CAD), CecropinA(1–11)–CecropinD(12–37); RP-HPLC, reversed-phase high liquid chromatography; CD, circular dichroism; IPTG, Isopropyl-β-d-thiogalactoside; ESI-MS, electrospray ionization mass spectrometry; SDS-PAGE, sodium dodecyl sulfate-polyac
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Expression and characterization of antimicrobial peptide CecropinAD in the methylotrophic yeast Pichia pastoris
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 44, Issue 1, January 2009, Pages 11–16
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us