Key Laboratory of Tropical Biological Resources, Ministry of Education, Hainan University, Haikou, Hainan, 570228, China.
Appl Microbiol Biotechnol. 2013 Feb;97(3):1223-30. doi: 10.1007/s00253-012-4287-6. Epub 2012 Jul 24.
Conotoxins are a diverse array of small peptides mostly with multiple disulfide bridges. These peptides become an increasing significant source of neuro-pharmacological probes and drugs as a result of the high selectivity for ion channels and receptors. Conotoxin GeXIVAWT (CTX-GeXIVAWT) is a 28-amino acid peptide containing five cysteines isolated from the venom of Conus generalis. Here, we present a simple and fast strategy of producing disulfide-rich conotoxins via recombinant expression. The codes of novel conotoxin gene GeXIVAWT were optimized and generated two pairs of primers by chemical synthesis for construction of expression vector. Recombinant expression vector pET22b(+)-GeXIVAWT fused with pelB leader and His-tag was successfully expressed as an insoluble body in Escherichia coli BL21(DE3) cells. Recombinant conotoxin GeXIVAWT (rCTX-GeXIVAWT) was obtained by dissolving the insoluble bodies and purifying with a Ni-NTA affinity column, which was further purified using reverse-phase high-performance liquid chromatography and identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The rCTX-GeXIVAWT renatured in vitro could inhibited the growth of Sf9 cell with biological activity assay. This expression system may prove valuable for future structure-function studies of conotoxins.
短肽毒素是一类具有多个二硫键的小分子多肽，主要来源于海螺。由于对离子通道和受体具有高度的选择性，短肽毒素已成为神经药理学探针和药物的重要来源。CTX-GeXIVAWT 是一种 28 个氨基酸的多肽，包含 5 个半胱氨酸残基，从海螺毒液中分离得到。本研究提出了一种通过重组表达生产富含二硫键短肽毒素的简单、快速策略。我们对新型短肽毒素基因 GeXIVAWT 进行了密码子优化，并通过化学合成设计了两对引物，用于构建表达载体。带有 pelB 信号肽和 His 标签的重组表达载体 pET22b(+)-GeXIVAWT 在大肠杆菌 BL21(DE3)细胞中以不溶性包涵体的形式表达。通过溶解包涵体并使用 Ni-NTA 亲和柱纯化，获得重组短肽毒素 GeXIVAWT(rCTX-GeXIVAWT)，然后进一步使用反相高效液相色谱法纯化，并通过基质辅助激光解吸/电离飞行时间质谱进行鉴定。体外复性的 rCTX-GeXIVAWT 具有生物活性，可抑制 Sf9 细胞的生长。该表达系统可能对未来短肽毒素的结构功能研究具有重要价值。
