Expression and purification of recombinant human α-defensins in Escherichia coli
Expression and purification of recombinant human α-defensins in Escherichia coli
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DOI：
10.1016/j.pep.2006.05.004
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发表时间：
2006-09-01
影响因子：
1.6
通讯作者：
Lubkowski, Jacek
中科院分区：
文献类型：
--
作者：
Pazgier, Marzena;Lubkowski, Jacek
已经开发了不同的策略来使用重组技术生产小抗微生物肽（AMP）。到目前为止，获得大量活性重组人α-防御素的所有努力都只是中等成功。在这里，我们报告了一种有效的方法，生物合成的人α-防御素（hNP-1到hNP-3和hD-5和hD-6）在大肠杆菌。所有的肽，表达为与由E. coli色氨酸操纵子（trp Delta LE 1413多肽），通过固定化金属亲和层析（IMAC）从包涵体中分离，并通过化学切割与融合前导序列分离。根据简单的方案纯化的完全还原的肽随后被折叠、氧化，并进行功能和结构分析。除hD-6外，所有重组α-防御素均表现出预期的抗E。大肠杆菌活性，如通过殖民地计数方法测量的。本报告中描述的方法是一种低成本、有效的方法，可以产生从毫克到克的α-防御素。(c)2006年爱思唯尔公司All rights reserved.
Different strategies have been developed to produce small antimicrobial peptides (AMPs) using recombinant techniques. Up to now, all efforts to obtain larger quantities of active recombinant human alpha-defensins have been only moderately successful. Here we report an effective method of biosynthesis of human alpha-defensins (hNP-1 to hNP-3 and hD-5 and hD-6) in the Escherichia coli. All the peptides, expressed as insoluble fusions with the peptide encoded by a portion of E. coli tryptophan operon (trp Delta LE 1413 polypeptide), were isolated from the inclusion bodies by immobilized metal affinity chromatography (IMAC) and separated from the fusion leader by chemical cleavage. Fully reduced peptides that were purified according to a straightforward protocol were subsequently folded, oxidized, and subjected to functional and structural analyses. With the exception of hD-6, all recombinant alpha-defensins exhibit expected anti-E. coli activity, as measured by the colony counting method. The method described in this report is a low-cost, efficient way of generating alpha-defensins in quantities ranging from milligrams to grams. (c) 2006 Elsevier Inc. All rights reserved.