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首页> 外文期刊>Protein Expression and Purification >Bacterial production of latarcin 2a, a potent antimicrobial peptide from spider venom
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Bacterial production of latarcin 2a, a potent antimicrobial peptide from spider venom

机译：细菌毒Latarcin 2a的细菌生产

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Natural venoms are promising sources of candidate therapeutics including antibiotics. A recently described potent antimicrobial peptide latarcin 2a (Ltc 2a) from Lachesana tarabaevi spider venom shows a broad-spectrum antibacterial activity. This peptide consists of 26 amino acid residues and therefore its production using chemical synthesis, although trivial, is costly. We describe an easy approach to Ltc 2a production in Escherichia coli using the conventional fusion partner thioredoxin. Latarcin 2a synthetic gene was cloned into the expression vector pET-32b, which was then used to transform E. coli BL21(DE3) strain. His-tagged fusion purification was achieved using metal-chelate affinity chromatography. Since no methionine residues are present in the latarcin 2a sequence, cyanogen bromide could be effectively utilized to separate the target product from the carrier protein. Reverse-phase HPLC was used as the final step of purification; the final yield was similar to 3 mg/L of bacterial culture. To increase the yields, we attempted incorporation of Ltc 2a tandem repeats into the fusion protein; however, production rates greatly decreased due to enhanced fusion toxicity. Moreover, we probed constructs to produce an Ltc 2a dimer and the Ltc 2a propeptide to study their functional properties. Recombinant peptides were produced at appreciable yields and biological tests to determine their activities were performed. Latarcin 2a is the first linear peptide from spider venom and one of the first membrane-active peptides from venomous animals to be biosynthetically produced. (c) 2008 Elsevier Inc. All rights reserved.
机译：天然毒液是包括抗生素在内的候选疗法的有前途的来源。最近描述的来自Lachesana tarabaevi蜘蛛毒液的有效抗菌肽latarcin 2a（Ltc 2a）显示了广谱抗菌活性。该肽由26个氨基酸残基组成，因此使用化学合成方法生产虽然很琐碎，但价格昂贵。我们描述了一种使用常规融合伴侣硫氧还蛋白在大肠杆菌中生产Ltc 2a的简便方法。将Latarcin 2a合成基因克隆到表达载体pET-32b中，然后将其用于转化大肠杆菌BL21（DE3）菌株。使用金属螯合物亲和色谱可实现带有组氨酸标签的融合纯化。由于在larartin 2a序列中不存在甲硫氨酸残基，因此可以有效地利用溴化氰从载体蛋白中分离目标产物。反相HPLC用作纯化的最终步骤;最终产量类似于3 mg / L的细菌培养物。为了提高产量，我们尝试将Ltc 2a串联重复序列掺入融合蛋白中。但是，由于融合毒性增强，生产率大大降低。此外，我们探查了产生Ltc 2a二聚体和Ltc 2a前肽的构建体，以研究其功能特性。以可观的产量产生重组肽，并进行生物学测试以确定其活性。 Latarcin 2a是来自蜘蛛毒液的第一个线性肽，也是来自生物合成的有毒动物的第一个膜活性肽之一。 （c）2008 Elsevier Inc.保留所有权利。

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