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

摘要:

STK11蛋白（serine/threonine kinase 11）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。

Abstract:

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-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.