To establish a simple, quick and effective method to get a large amount of spider toxin JZTX-26 (35 aa) and JZTX-51 (27 aa) with 3 disulfide bonds each, the mature peptides coding gene fragments were constructed and fused with maltose-binding protein (MBP) tag in an Escherichia coli expression vector pMAL-p2x. The recombinant constructs pMAL-jz26 and pMAL-jz51 were transformed and cultured in E. coli TB1 and BL21 (DE3). After being induced by isopropyl-β-d-thiogalactoside (IPTG), the periplasmic proteins were purified by amylose affinity chromatography and analyzed by SDS-PAGE. The fusion proteins were digested with factor X, and purified by Sizes-Exclusion chromatography and Reversed Phase HPLC. Molecular weights of the purified peptides were obtained by using a MALDI-TOF-TOF mass spectrometer, which were consistent with the theoretical molecular weights. Five milligram of target protein could be purified from 1 L of culture medium. The results indicate that the peptides with three disulfide bonds can be expressed by using the prokaryotic expression system with MBP tag. Our findings suggest the possibility of genetic engineering to obtain large amount of spider peptide toxins.
为建立一种简便、快速且能大量获得富含二硫键的蜘蛛多肽毒素JZTX-26 (35 aa) 和JZTX-51 (27 aa) 的有效方法，利用PCR 的方法克隆成熟肽编码基因并插入至大肠杆菌Escherichia coli 表达载体pMAL-p2x 中与MBP(麦芽糖结合蛋白) 标签融合，构建重组表达质粒pMAL-jz26 和pMAL-jz51。在受体菌TB1 和BL21 (DE3) 中对两个重组表达质粒分别进行IPTG 诱导表达，通过Amylose 亲和层析柱纯化并进行SDS-PAGE 分析；采用因子X对融合蛋白进行酶切后通过分子筛以及反相高效液相色谱对两种重组蛋白进行纯化。通过MALDI-TOF-TOF 质谱鉴定，表达产物的分子量与预期的多肽理论分子量一致。1 L 表达培养液中能获得大约5 mg 纯化的目的蛋白JZTX-26 或JZTX-51。结果表明利用该原核表达体系可对蜘蛛毒素基因jztx-26 和jztx-51 进行融合表达，并对重组蛋白进行亲和层析，为采用基因工程的手段大量获得蜘蛛多肽毒素奠定了基础。.
Keywords: DRG cell; fusion protein; prokaryotic expression technology; sodium channel; spider toxin.