Hou Xin, Hu Ting-mao, Liu Jun-e
College of Life Sciences, Inner Mongolia University, Hohhot 010021, China.
Wei Sheng Wu Xue Bao. 2007 Feb;47(1):79-82.
STK11 (serine/threonine kinase 11 ), a multi-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 cDNA 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-STK 1 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.
STK11（丝氨酸/苏氨酸激酶11）是最近报道的一种多功能蛋白，可能参与广泛的细胞过程，包括细胞周期调控、p53介导的细胞凋亡、ras诱导的细胞转化和细胞极化。在大肠杆菌中高效表达功能性STK11将促进对其结构和功能的研究。用pET-44a(+)和我们实验室克隆的STK11基因的cDNA构建了诱导型原核表达载体pET-Nus-STK11（带有Nus融合标签）。然后在IPTG诱导下，pET-Nus-STK11在BL21(DE3)和Rosetta-gami(DE3)pLysS中表达。SDS-PAGE和Western印迹表明，在BL21(DE3)中获得的重组Nus-STK11以包涵体形式存在，而来自Rosetta-gami(DE3)pLysS的主要存在于可溶部分，分别占总蛋白的8.9%和16.7%。纯化和复性后，将获得的重组蛋白通过Chariot导入SMMC-7721细胞，观察其对细胞生长和细胞周期的影响。已证明来自BL21(DE3)的Nus-STK 1没有任何肿瘤抑制活性，而观察到对SMMC-7721细胞的生长抑制率为47.05%，并且来自Rosetta-gami(DE3)pLysS的Nus-STK11也使SMMC-7721细胞的细胞周期进程从G0/G1期阻滞到S期，表明上述来自Rosetta-gami(DE3)pLysS的重组融合蛋白具有显著的生物学活性。这是关于在大肠杆菌中表达功能性重组STK11蛋白的首次报道。
