Abstract

Opiorphin secreted into the human saliva is an endogenous regulator pentapeptide (QRFSR) showing a pain suppressant effect similar to morphine. We report here a strategy of producing tandemly repeated multimers of opiorphin peptide efficiently by expressing in Esherichia coli cells. For improving the expression level and increasing the yield of pentapeptide, 32-mer tandem sequence of opiorphin was generated by using partially complementary primers inserted into pGEX-2T expression vector, following fused with glutathione transferase (GST) gene. Recombinant tandem multimer was easily captured by GST column with a yield of 9.2 mg/L, and then 32-mer opiorphin polypeptide of 28.49 kDa size was cleaved into monomers by CNBr and endoproteinase Asp-N. Opiorphin polypeptides having a molecular weight of about 693 Da and containing 5 amino acids in length were then generated. The amount of cleaved and purified recombinant opiorphin monomers was calculated as 6.2 mg/L by the Bradford assay. Protein expression and cleavage of recombinant polypeptide were confirmed by SDS-PAGE and TLC. Results from this study clearly open a door to the mass production of the opiorphin peptide to use for laboratory research and industrial purposes. The overall gene construction and the direct cloning to an expression vector strategy using partially complementary primers with cleavable linker peptides could be applicable to many small peptides and their analogs for pharmaceutical and clinical application.

中文翻译：

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通过合理设计引物重组生产卵磷脂五肽串联多聚体

摘要

分泌到人唾液中的卵磷脂是一种内源性调节剂五肽（QRFSR），具有类似于吗啡的止痛作用。我们在这里报告了一种通过在大肠杆菌中表达来有效产生串联重复的opiorphin肽多聚体的策略细胞。为了提高表达水平并增加五肽的产量，通过将部分互补的引物与谷胱甘肽转移酶（GST）基因融合，使用插入pGEX-2T表达载体中的部分互补引物，生成了32联体的opiorphin序列。重组串联多聚体易于通过GST色谱柱捕获，产量为9.2 mg / L，然后通过CNBr和内切蛋白酶Asp-N将28.49 kDa大小的32-mer opiorphin多肽裂解为单体。然后产生具有约693Da的分子量并且长度为5个氨基酸的卵磷脂多肽。通过Bradford测定，切割和纯化的重组opiorphin单体的量为6.2mg / L。通过SDS-PAGE和TLC证实重组多肽的蛋白质表达和切割。这项研究的结果显然为鸦片蛋白肽的大规模生产打开了一扇门，可用于实验室研究和工业用途。使用具有可裂解的接头肽的部分互补引物的整体基因构建和直接克隆至表达载体的策略可适用于许多小肽及其类似物，用于药物和临床应用。