期刊论文

Recombinant expression and purification of spider toxin, JZTX-51 and JZTX-26, from Chilobrachys jingzhao

Chen, J.

中文

生物工程学报

1000-3061

2018

34

10

1668-1678

10.13345/j.cjb.170529

Nos.31672457:国家自然科学基金 31772865:国家自然科学基金 No.16A098:湖南省教育厅重点项目 No.2017NK2311:湖南省科技厅重点项目 No.XCX17071:湖南农业大学大学生创新项目

本校为第一且通讯机构

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。1L表达培养液中能获得大约5mg纯化的目...

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 chrom...