High-level expression of an antimicrobial peptide histonin as a natural form by multimerization and furin-mediated cleavage
High-level expression of an antimicrobial peptide histonin as a natural form by multimerization and furin-mediated cleavage
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DOI：
10.1007/s00253-007-1273-5
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发表时间：
2008-02-01
影响因子：
5
通讯作者：
Kim, Sun Chang
中科院分区：
文献类型：
--
作者：
Kim, Jung Min;Jang, Su A.;Kim, Sun Chang
抗菌肽（AMP）在大肠杆菌中的直接表达引起几个问题，例如AMP对宿主细胞的毒性，其对蛋白水解降解的敏感性，以及由于AMP从融合配偶体切割后引入的额外残基而导致的抗微生物活性降低。为了克服这些问题，在大肠杆菌中大量生产一种有效的AMP组蛋白（RAGLQFPVGKLLKKLLKRLKR）。在大肠杆菌中，建立了一个高效的表达系统，在该系统中，组蛋白的毒性被融合伴侣F4（PurF蛋白的截短片段）中和，并且通过组蛋白基因的多聚体表达来提高产量。融合蛋白的表达水平在组蛋白基因的12聚体时达到最大。此外，由于组蛋白C末端存在RLKR残基，弗林蛋白酶切割表达的多聚体组蛋白产生完整的天然组蛋白。结果表明，E.大肠杆菌中的表达与合成的组蛋白相同。用我们的表达系统，从1升E.大肠杆菌培养。这些结果可能导致用于大规模生产对宿主有毒的AMP的具有成本效益的解决方案。
Direct expression of an antimicrobial peptide (AMP) in Escherichia coli causes several problems such as the toxicity of AMP to the host cell, its susceptibility to proteolytic degradation, and decreased antimicrobial activity due to the additional residue(s) introduced after cleavage of AMPs from fusion partners. To overcome these problems and produce a large quantity of a potent AMP histonin (RAGLQFPVGKLLKKLLKRLKR) in E. coli, an efficient expression system was developed, in which the toxicity of histonin was neutralized by a fusion partner F4 (a truncated fragment of PurF protein) and the productivity was increased by a multimeric expression of a histonin gene. The expression level of the fusion proteins reached a maximum with a 12-mer of a histonin gene. In addition, because of the RLKR residues present at the C terminus of histonin, furin cleavage of the multimeric histonin expressed produces an intact, natural histonin. The AMP activity of the histonin produced in E. coli was identical to that of a synthetic histonin. With our expression system, 167 mg of histonin was obtained from 1 l of E. coli culture. These results may lead to a cost-effective solution for the mass production of AMPs that are toxic to a host.