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

为了获得有活性的白喉毒素突变体蛋白(Cross-reactingmaterial197,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16h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。

Inhibition of cystathionine β-synthase promotes apoptosis and reduces cell proliferation in chronic myeloid leukemia

Increased endogenous hydrogen sulfide(H_(2)S)level by cystathionine β-synthase(CBS)has been shown to closely relate tumorigenesis.H_(2)S promotes angiogenesis,stimulates bioenergy metabolism and inhibits selective phosphatases.However,the role of CBS and H_(2)S in chronic myeloid leukemia(CML)remains elusive.In this study,we found that CBS and H_(2)S levels were increased in the bone marrow mononuclear cells of pediatric CML patients,as well as in the CML-derived K562 cells and CBS expression levels were correlated with different disease phases.Inhibition of CBS reduced the proliferation of the CML primary bone marrow mononuclear cells and induced growth inhibition,apoptosis,cell cycle arrest,and migration suppression in K562 cells and tumor xenografts.The knockdown of CBS expression by shRNA and inhibiting CBS activity by AOAA decreased the endogenous H_(2)S levels,promoted mitochondrial-related apoptosis and inhibited the NF-xB-mediated gene expression.Our study suggests that inhibition of CBS induces cell apoptosis,as well as limits cell proliferation and migration,a potential target for the treatment of chronic myeloid leukemia.