[Expression of functional human STK11 protein in Escherichia coli].
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.
Hou
Xin
X
College of Life Sciences, Inner Mongolia University, Hohhot 010021, China. houxinliu@yahoo.com.cn
Hu
Ting-mao
TM
Liu
Jun-e
JE
D000091162:AMP-Activated Protein Kinase Kinases; D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000494:pharmacology
D011993:Recombinant Fusion Proteins; D017346:Protein Serine-Threonine Kinases; C109790:STK11 protein, human; D000091162:AMP-Activated Protein Kinase Kinases