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首页> 外文期刊>Protein Expression and Purification >Bacterial production and purification of SGPI-1 and SGPI-2, two peptidic serine protease inhibitors from the desert locust, Schistocerca gregaria
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Bacterial production and purification of SGPI-1 and SGPI-2, two peptidic serine protease inhibitors from the desert locust, Schistocerca gregaria

机译：来自沙漠蝗虫血吸虫的两种肽丝氨酸蛋白酶抑制剂SGPI-1和SGPI-2的细菌生产和纯化

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摘要

The last decade, a new scrine protease inhibitor family has been described in arthropods. Eight members were purified from the locusts Locusta migratoria (LMPI-1-2 and HI) and Schistocerca gregaria (SGPI-1-5) and 11 additional locust peptides were identified by cDNA cloning. Furthermore, the light chain of the 155-kDa heterodimeric protease inhibitor pacifastin, from the freshwater crayfish Pacifastacus leniusculus, was found to be composed of nine consecutive inhibitory domains (PLDs). These domains share a pattern of 6 conserved cysteine residues (Cys-Xaa(9-12)-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa(2-3)-Gly-Xaa(3-4)-Cys-Thr-Xaa(3 )-Cys) with the locust inhibitors. So far, for most of the PLD-related peptides the biological functions remain obscure. To obtain sufficient amounts of material to perform physiological experiments, we have optimised the production of SGPI-1-2 via a bacterial (Escherichia coli) expression system. The cDNA sequences encoding these peptides were inserted in the pMAL-2pX vector, downstream of the gene encoding the maltose-binding protein (including a signal peptide). As a consequence, both peptides were expressed as fusion proteins (2-3 mg/l) and targeted to the periplasmic space. Following a one-step affinity purification, both fusion proteins were successfully cleaved by Factor Xa and after a methanol extraction, it took only one additional RP-HPLC run to purify both peptides to homogeneity. Finally, the formation of the disulphide bridges and the biological activity of the recombinant peptides were verified by mass spectrometry and a spectrophotometric protease inhibitor assay, respectively. (C) 2003 Elsevier Science (USA). All rights reserved. [References: 24]
机译：在最近的十年中，节肢动物中已经描述了新的神经蛋白酶蛋白酶抑制剂家族。从蝗虫Locusta migratoria（LMPI-1-2和HI）和Schistocerca gregaria（SGPI-1-5）中纯化了八种成员，并通过cDNA克隆鉴定了另外11种蝗虫肽。此外，发现淡水小龙虾Pacifastacus leniusculus的155 kDa异二聚体蛋白酶抑制剂pacifastin的轻链由九个连续的抑制域（PLD）组成。这些域共有6个保守的半胱氨酸残基（Cys-Xaa（9-12）-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa（2-3）-Gly-Xaa（3-4）-Cys- Thr-Xaa（3）-Cys）与蝗虫抑制剂。迄今为止，对于大多数PLD相关肽，其生物学功能仍然不清楚。为了获得足够数量的材料来进行生理学实验，我们通过细菌（大肠杆菌）表达系统优化了SGPI-1-2的生产。将编码这些肽的cDNA序列插入到编码麦芽糖结合蛋白（包括信号肽）的基因的下游的pMAL-2pX载体中。结果，两种肽均表达为融合蛋白（2-3 mg / l），并靶向周质空间。一步亲和纯化后，两个融合蛋白均成功被因子Xa裂解，甲醇提取后，仅需再进行一次RP-HPLC纯化，即可将两种肽纯化至均一。最后，分别通过质谱法和分光光度蛋白酶抑制剂测定法验证了二硫键的形成和重组肽的生物学活性。 （C）2003 Elsevier Science（美国）。版权所有。 [参考：24]

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