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

文献类型： 外文期刊

第一作者： Yang, Yalin

作者： Yang, Yalin;Tian, Zigang;Teng, Da;Zhang, Jun;Wang, Jiarong;Wang, Jianhua

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期刊名称：JOURNAL OF BIOTECHNOLOGY （ 影响因子：3.307； 五年影响因子：3.778 ）

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收录情况： SCI

摘要： 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 (~20kDa) 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 ~10kDa protein, was found. Compared with the unknown site, the specific site of IEGR[downward arrow]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.

分类号： Q81

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