分子伴侣对白喉毒素无毒突变体CRM197重组蛋白在大肠杆菌中可溶性表达的促进作用

Molecular chaperones facilitate soluble expression of recombinant non-toxic mutant CRM197 of diphtheria toxin in Escherichia coli

摘要:

为了获得有活性的白喉毒素突变体蛋白 (Cross-reacting material 197，CRM197)，本研究利用分子伴侣pG-KJE8与重组质粒pET28a-CRM197在大肠杆菌原核表达系统中进行共表达，来促进目的蛋白的正确折叠，进而提高CRM197蛋白的可溶性表达。将质粒转化至大肠杆菌后并诱导其表达目的蛋白，再通过SDS-PAGE胶染色、Western blotting等技术对所得蛋白进行检测分析。结果发现：利用体外重组技术成功得到了pET28a-CRM197重组蛋白原核表达质粒，且CRM197重组蛋白在原核表达系统中主要以包涵体形式表达；通过探索和优化，确定了诱导蛋白的最佳浓度和温度，当加入终浓度为1.0 mmol/L IPTG、0.5 mg/mL L-阿拉伯糖、5.0 ng/mL四环素,在20 ℃条件下诱导16 h时，目的蛋白的可溶性表达得到显著提高；可溶性表达的CRM197重组蛋白可以与CRM197一抗发生特异性结合，免疫反应性良好。因此，研究发现分子伴侣pG-KJE8可以促进CRM197重组蛋白在大肠杆菌中以可溶性形式表达，且能很好地与CRM197一抗发生特异性结合，证实CRM197重组蛋白具有良好的免疫反应性，为CRM197蛋白的工业化生产及应用奠定了一定的基础。

Abstract:

Diphtheria toxin is an ADP-ribosyltransferase toxic to human cells. Mutation of the active site in its catalytic domain eliminates the toxicity, but retains its immunogenicity. A non-toxic mutant of diphtheria toxin known as CRM197 protein has become an ideal carrier protein for conjugate vaccines. CRM197 can further improve its immunogenicity by cross-linking with other antigens, so it has good potential to find broad applications. Unfortunately, inclusion bodies are easily formed during the expression of recombinant CRM197 protein in Escherichia coli, which greatly reduces its yield. In order to address this problem, pG-KJE8 vector carrying molecular chaperones and plasmid pET28a-CRM197, were co-expressed in Escherichia coli. The results showed that the recombinant CRM197 protein was successfully expressed and appeared largely in inclusion bodies. The molecular chaperones DnaK, DnaJ, GrpE, GroES and GroEL5 expressed can facilitate correct and rapid folding of CRM197. Furthermore, it can also improve the recovery rate of soluble CRM197 protein. The soluble expression of CRM197 was maximized upon addition of 1.0 mmol/L IPTG, 0.5 mg l-arabinose, 5.0 ng/mL tetracycline and induction at 20oC for 16 h. The soluble CRM197 protein shows good immunoreactivity, demonstrating the molecular chaperones expressed from pG-KJE8 facilitated the soluble expression of CRM197 protein in E. coli.