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High-level production of a candidacidal peptide lactoferrampin in Escherichia coli by fusion expression

Paper ID Volume ID Publish Year Pages File Format Full-Text
24746 43534 2009 6 PDF Available
Title
High-level production of a candidacidal peptide lactoferrampin in Escherichia coli by fusion expression
Abstract

Expression of lactoferrampin 265–284 (Lfampin20), a potential candidacidal agent with 20 amino acid segment from lactoferrin, in Escherichia coli was explored. The DNA fragment encoding Lfampin20 was synthesized in light of the E. coli preferred codons by “partially overlapping primer-based PCR” method. The Lfampin20 gene was fused with thioredoxin (Trx) gene to construct a recombinant plasmid pETLfa20. The resulting expression level of the fusion protein Trx-Lfampin20 (∼20 kDa) accounted for 34–42% of cellular protein, and about 52% of the target proteins were in a soluble form. Soluble Trx-Lfampin20 accounted for 66% of the total soluble proteins. The soluble fusion protein was easily purified to near homogeneity by affinity chromatography using hexahistidine tag. Recombinant Lfampin20 was effectively obtained by on-column cleavage of the fusion protein with factor Xa. An unknown site in the Trx-tag fusion protein, which can be cleaved by factor Xa to produce ∼10 kDa protein, was found. Compared with the unknown site, the specific site of IEGR↓X was easier to be recognized and cleaved by factor Xa. The molecular mass of recombinant Lfampin20 determined by MALDI-TOF (matrix assisted laser desorption ionization-time-of-flight) is equal to its theoretical molecular weight. Antimicrobial activity assays demonstrated that the recombinant Lfampin20 had candidacidal activity. Integration of the key strategies for the expression of antimicrobial peptides (AMPs) such as codon usage bias, fusion partner and on-column cleavage, would provide an efficient and facile platform for the production or study of AMPs.

Keywords
AMPs, antimicrobial peptides; IPTG, isopropyl-1-thio-β-d-galactopyranoside; Lfampin20, Lactoferrampin 265–284; C. albicans, Candida albicans; E. coli, Escherichia coliFusion expression; Candidacidal peptide; Lactoferrampin; Escherichia coli; On-column cle
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High-level production of a candidacidal peptide lactoferrampin in Escherichia coli by fusion expression
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 139, Issue 4, 23 February 2009, Pages 326–331
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