Expression, purification and characterization of cecropin antibacterial peptide from Bombyx mori in Saccharomyces cerevisiae
Expression, purification and characterization of cecropin antibacterial peptide from Bombyx mori in Saccharomyces cerevisiae
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DOI：
10.1016/j.pep.2013.02.013
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发表时间：
2013-07-01
影响因子：
1.6
通讯作者：
Zhang, Fuchun
中科院分区：
文献类型：
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作者：
Xia, Lijie;Liu, Zhongyuan;Zhang, Fuchun
CecropinXJ是从家蚕幼虫中分离得到的一种阳离子抗菌肽。本研究将抗菌肽基因cecropinXJ克隆到pYES2/CT/alpha，因子表达载体中，在酿酒酵母INVSc1菌株中表达。重组蛋白在酵母中诱导表达120 h后，最大总分泌蛋白量为1.437 g/L。重组cecropinXJ蛋白占总蛋白的79.45%。重组物cecropinXJ经Ni-NTA琼脂糖柱纯化后，对革兰氏阴性菌和革兰氏阳性菌均有较强的抑菌活性。其对大肠杆菌ATCC25922的最小抑制浓度（MIC）为0.81 μ m，透射电镜（TEM）分析表明，与未处理的对照组相比，处理后的病原菌表面发生了明显的形态学变化，表明该抗菌肽通过直接破坏微生物膜发挥作用。即使肽浓度为200 μ m， CecropinXJ对红细胞的溶血作用也很小，因此，CecropinXJ选择性地作用于细菌膜。纯化的重组抗菌肽cecropinXJ在4 ~ 100℃的温度范围和pH 2.0 ~ 12.0的pH范围内对大肠杆菌ATCC25922保持了较高的稳定性。综上所述，本研究表明重组cecropinXJ可以通过基因工程方法在酵母中大量生产，并且对所有被试微生物都具有强而快速的抑菌活性。我们的结果表明cecropinXJ是一种潜在的治疗候选药物。(C) 2013爱思唯尔公司版权所有。
CecropinXJ is a cationic antimicrobial peptide originally isolated from the larvae of Bombyx mori. In this study, an antibacterial peptide gene of cecropinXJ was cloned into the pYES2/CT/alpha, Factor expression vector and expressed in the Saccharomyces cerevisiae INVSc1 strain. Following an induction of recombinant protein expression in yeast for 120 h, the maximum amount of total secreted protein was 1.437 g/L. The percentage of recombinant cecropinXJ was estimated to be 79.45% of the total protein. After purification with Ni-NTA agarose column, recombinant cecropinXJ was noted to exert strong antimicrobial activities against a broad-spectrum of microorganisms, including Gram-negative and Gram-positive bacteria. Its minimal inhibitory concentration (MIC) against Escherichia coli ATCC25922 was 0.81 mu M. In addition, transmission electron microscopy (TEM) analysis indicated that the surfaces of the treated pathogens underwent obvious morphological changes compared with the untreated controls, suggesting that this antimicrobial peptide exerts its action by directly disrupting membranes of microorganisms. CecropinXJ had a small hemolytic effect on red blood cells even with a peptide concentration of 200 mu M. Thus, cecropinXJ acts selectively on bacterial membranes. Purified recombinant antibacterial peptide, cecropinXJ, retained a high stability against E. coli ATCC25922 over a temperature range from 4 degrees C to 100 degrees C and a pH range from pH 2.0 to 12.0. Taken together, this study demonstrates that recombinant cecropinXJ can be produced in large quantities in yeast with genetic engineering methods, and that it has strong and rapid antimicrobial activities against all of microorganisms tested. Our results suggest that cecropinXJ is a potential candidate for therapy. (C) 2013 Elsevier Inc. All rights reserved.