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[Expression of functional human STK11 protein in Escherichia coli]
Xin Hou1, Ting-mao Hu, Jun-e Liu
1College of Life Sciences, Inner Mongolia University, Hohhot 010021, China. houxinliu@yahoo.com.cn
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-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.
Insights
This study successfully expressed functional serine/threonine kinase 11 (STK11) in Escherichia coli. The recombinant STK11 protein demonstrated significant biological activity, inhibiting SMMC-7721 cell growth and arresting cell cycle progression.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Serine/threonine kinase 11 (STK11) is a multifunctional protein involved in critical cellular processes like cell cycle regulation and apoptosis.
- Efficient expression of functional STK11 in prokaryotic systems is crucial for further structural and functional studies.
- Previous attempts at expressing STK11 in Escherichia coli have faced challenges in achieving soluble and active protein.
Purpose of the Study:
- To construct an inducible prokaryotic expression vector for STK11.
- To efficiently express functional recombinant STK11 protein in Escherichia coli.
- To evaluate the biological activity of the expressed STK11 protein in SMMC-7721 cells.
Main Methods:
- Construction of the pET-Nus-STK11 expression vector.
- Expression of recombinant STK11 in BL21 (DE3) and Rosetta-gami (DE3)pLysS E. coli strains.
- Analysis of protein expression using SDS-PAGE and Western blot.
- Purification, refolding, and cellular uptake of recombinant STK11 using Chariot.
- Assessment of cell growth inhibition and cell cycle arrest in SMMC-7721 cells.
Main Results:
- Recombinant STK11 was expressed in both E. coli strains, with Rosetta-gami (DE3)pLysS yielding a higher proportion of soluble protein (16.7%) compared to BL21 (DE3) (8.9%).
- STK11 expressed in BL21 (DE3) showed no tumor-suppression activity.
- Recombinant STK11 from Rosetta-gami (DE3)pLysS exhibited significant biological activity, inhibiting SMMC-7721 cell growth by 47.05% and causing G0/G1 to S phase cell cycle arrest.
Conclusions:
- Rosetta-gami (DE3)pLysS is a suitable host for the soluble and functional expression of recombinant STK11 in E. coli.
- The expressed recombinant STK11 protein possesses significant tumor-suppression activity.
- This study reports the first successful expression of functional recombinant STK11 protein in Escherichia coli.
