Skosyrev Vitaly S, Kulesskiy Evgeny A, Yakhnin Alexander V, Temirov Yuri V, Vinokurov Leonid M
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Pushchino Branch, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia.
Protein Expr Purif. 2003 Apr;28(2):350-6. doi: 10.1016/s1046-5928(02)00697-6.
Sarcotoxin IA is an antibacterial peptide that is secreted by a meat-fly Sarcophaga peregrina larva in response to a hypodermic injury or bacterial infection. This peptide is highly toxic against a broad spectrum of both Gram-positive and Gram-negative bacteria and lethal to microbes even at nanomolar concentrations. However, research needs as well as its potential use in medicine require substantial amounts of highly purified sarcotoxin. Because heterologous expression systems proved to be inefficient due to sarcotoxin sensitivity to intracellular proteases, here we propose the biosynthesis of sarcotoxin precursors in Escherichia coli cells that are highly sensitive to the mature peptide. To optimize its biosynthesis, sarcotoxin was translationally fused with proteins highly expressed in E. coli. A fusion partner and the position of sarcotoxin in the chimeric polypeptide were crucial for protecting the sarcotoxin portion of the fusion protein from proteolysis. Released after chemical cleavage of the fusion protein and purified to homogeneity, sarcotoxin displayed antibacterial activity comparable to that previously reported for the natural peptide.
肌毒素IA是一种抗菌肽，由肉蝇（Sarcophaga peregrina）幼虫在受到皮下损伤或细菌感染时分泌。这种肽对革兰氏阳性菌和革兰氏阴性菌均具有高度毒性，即使在纳摩尔浓度下对微生物也具有致死性。然而，研究需求及其在医学上的潜在用途需要大量高度纯化的肌毒素。由于肌毒素对细胞内蛋白酶敏感，异源表达系统效率低下，因此我们在此提出在对成熟肽高度敏感的大肠杆菌细胞中生物合成肌毒素前体。为了优化其生物合成，将肌毒素与在大肠杆菌中高表达的蛋白质进行翻译融合。融合伴侣以及嵌合多肽中肌毒素的位置对于保护融合蛋白的肌毒素部分不被蛋白酶水解至关重要。融合蛋白经化学裂解后释放并纯化至同质，肌毒素显示出与先前报道的天然肽相当的抗菌活性。
