在大肠杆菌中表达有活性的人STK11蛋白  

Expression of functional human STK11 protein in Escherichia coli

作　　者：侯鑫[1] 扈廷茂[1] 刘俊娥[2] 

机构地区：[1]内蒙古大学生命科学学院,呼和浩特010021 [2]中国科学院微生物研究所,北京100080

出　　处：《微生物学报》2007年第1期79-82,共4页Acta Microbiologica Sinica

基　　金：内蒙古教育厅重点研究项目(NJ02005)~~

摘　　要：STK11蛋白(serine/threonine kinase11)是近年来发现的具有多种重要功能的蛋白,可参与调控细胞周期、p53介导的细胞凋亡、ras诱导的细胞转化、细胞极化等多种生物学过程。利用大肠杆菌高效表达有活性的人STK11蛋白,可为其结构和功能的深入研究打下良好基础。利用本室克隆的人STK11 cDNA和原核表达载体pET-44a(+)构建带有Nus融合标签的诱导型表达载体pET-Nus-STK11,在不同的大肠杆菌宿主中诱导表达。SDS-PAGE和Western blot检测表明,在BL21(DE3)宿主中表达的融合蛋白主要以包涵体形式存在,占菌体总蛋白的8.9%;在Rosetta-gami(DE3)pLysS宿主中主要表达为可溶性蛋白,占菌体总蛋白的16.7%。而经纯化和包涵体蛋白复性处理后,以Chariot介导重组融合蛋白进入人肝癌细胞SMMC-7721检测其对细胞生长和细胞周期的影响。与对照组相比,BL21(DE3)中表达的Nus-STK11蛋白几乎无抑制活性;而Rosetta-gami(DE3)pLysS中表达的Nus-STK11蛋白可以显著抑制SMMC-7721细胞的生长,抑制率达47.05%,并导致细胞周期的G0/G1期阻滞,证实表达的重组融合蛋白具有明显的生物学活性。上述结果为在大肠杆菌中成功表达有活性的重组STK11蛋白的首次报道。STK11 （serine/threonine kinase 11 ）, a muhi-functional protein reported recently, possibly participates in a broad range of cellular processes, including regulation of cell cycle, p53-mediated apoptosis, ras-induced cell transformation and cell polarization. An efficient expression of functional STK11 in Escherichia coli will promote the study on its structure and function. Inducible prokaryotic expression vector pET-Nus-STK11 （with Nus fusion tag） was constructed with pET-44a（ ＋ ） and the eDNA of STK11 gene cloned in our lab. pET-Nus-STK11 was then expressed in both BL21 （DE3） and Rosetta-gami （DE3）pLysS on the induction of IPTG. SDS-PAGE and Western blot indicated that recombinant Nus-STK11 obtained in BL21 （DE3） was in the form of inclusion body, whereas that from Rosetta-gami （DE3）pLysS was mainly in soluble fraction, and accounted for 8.9% and 16.7% of the total protein, respectively. After purification and refolding, the obtained recombinant protein was carried into SMMC-7721 cells by Chariot to observe its influence on cell growth and cell cycle. Nus-STK11 from BL21 （DE3） was proved to be lack of any tumor-suppression activity, while a growth inhibitory ratio of 47.05 % on SMMC-7721 cell was observed, and cell cycle progression of SMMC-7721 cells was also arrested from G0/G1 to S phase, with the Nus-STK11 from Rosetta-gami （DE3） pLysS, indicating that the above recombinant fusion protein from Rosetta-gami （DE3）pLysS had significant biological activity. This is the first report on functional recombinant STK11 protein expressed in Escherichia coli.

关 键 词：STK11蛋白 原核表达 Rosetta-gami(DE3)pLysS 

分 类 号：Q786[生物学—分子生物学]