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| Soluble Expression of Human HPPCn Recombinant Protein and Detection of Its Proliferation Activity |
| LU Qing-shan1, QIAO Yuan-yuan2, LI Jin-feng3, WANG Yun-liang3, WANG Shan-shan3, SHI Cheng-he2, YANG Xiao-peng1, ZHANG Da-jin2 |
1. Department of Neurology, The Second Affiliated Hospital of Zhengzhou University, Zhengzhou 450001, China;
2. Center for Basic Medical Sciences, Navy General Hospital of Chinese PLA, Beijing 100048, China;
3. The 148th Hospital of Chinese PLA, Zibo 255300, China |
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Abstract HPPCn is a new hepatocyte stimulating factor, and it is of great significance to obtain high purity of HPPCn for studying the biological function of HPPCn. The expression of HPPCn in the recombinant plasmid PET-24a(+)/HPPCn was mainly in the form of inclusion body. Cold-shock expression vector pColdⅡand seamless cloning technology were used to obtain recombinant plasmid pColdⅡ/HPPCn. The induction conditions, such as temperature, time and molecular chaperones, were optimized respectively. The expression product was subsequently purified by nickel-ion column. The specificity of the recombinant protein was analyzed by Western blotting. SMMC7721 cells were stimulated by human HPPCn recombinant protein at the concentrations of 0, 10 and 100ng/ml, respectively. The contents of PCNA and Brdu were determined by immunocytochemistry. Human HPPCn recombinant protein could be solubly expressed in the new system. The purity of recombinant HPPCn protein was 94.88%. Stimulated at different concentrations of HPPCn, Brdu incorporation and the expression levels of PCNA increased in a dose-dependent manner. The human HPPCn recombinant protein was induced to soluble expression in the cold-shock expression system, and the product could promote the proliferation of SMMC7721 cells. It may serve as the foundation for further studies on the mechanism of HPPCn.
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Received: 21 July 2015
Published: 22 December 2015
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| [1] LaBrecque D R, Pesch L A. Preparation and partial characterization of hepatic regenerative stimulator substance (SS) from rat liver. J Physiol, 1975, 248(2):273-284.
[2] Cui C P, Zhang D J, Shi B X, et al. Isolation and functional identification of a novel human hepatic growth factor:Hepatopoietin Cn+. Hepatology, 2008, 47(3):986-995.
[3] Matsuoka K, Taoka M, Satozawa N, et al. A nuclear factor containing the leucine-rich repeats expressed in murine cerebellar neurons. Proc Natl Acad Sci U S A, 1994, 91(21):9670-9674.
[4] Adegbola O, Pasternack G R. Phosphorylated retinoblastoma protein complexes with pp32 and inhibits pp32-mediated apoptosis. J Biol Chem, 2005, 280(16):15497-15502.
[5] 魏苹,杜劭君,崔春萍,等.新的肝细胞生长因子HPPCn的表达、纯化及体外活性检测.中国生物工程杂志, 2007, 27(10):6-11. Wei P, Du S J, Cui C P, et al. Expression, purification and activity detection of the new hepatocyte growth factor HPPCn. China Biotechnology, 2007, 27(10):6-11.
[6] Thomas J G, Ayling A, Baneyx F. Molecular chaperones, folding catalysts, and the recovery of active recombinant proteins from E. Coli. To fold or to refold. Appl Biochem Biotechnol, 1997, 66(3):197-238.
[7] Qing G, Ma L, Khorchid A, et al. Cold-shock induced high-yield protein production in Escherichia coli. Nat Biotechnol, 2004, 22(7):877-882.
[8] Benoit R M, Wilhelm R N, Scherer-Becker D, et al. An improved method for fast, robust, and seamless integration of DNA fragments into multiple plasmids. Protein Expr Purif, 2006, 45(1):66-71.
[9] Chen J H, Zhang T, Ju C, et al. An integrated immunoproteomics and bioinformatics approach for the analysis of Schistosoma japonicum tegument proteins. J Proteomics, 2014, 98:289-299.
[10] Lu Q. Seamless cloning and gene fusion. Trends Biotechnol, 2005, 23(4):199-207.
[11] Derman A I, Prinz W A, Belin D, et al. Mutations that allow disulfide bond formation in the cytoplasm of Escherichia coli. Science, 1993, 262(5140):1744-1747.
[12] Ellis R J. The molecular chaperone concept. Semin Cell Biol, 1990, 1(1):1-9.
[13] Vasina J A, Baneyx F. Expression of aggregation-prone recombinant proteins at low temperatures:A comparative study of the Escherichia coli cspA and tac promoter systems. Protein Expr Purif, 1997, 9(2):211-218.
[14] 张达矜,崔春萍,乔媛媛,等.新的肝细胞生长因子HPPCn的人多组织表达谱分析.军事医学科学院院刊, 2010, 34(06):505-508. Zhang D J, Cui C P, Qiao Y Y, et al. Human multiple tissue expression profile analysis of new hepatocyte growth factor HPPCn. Bulletin of the Academy of Military Medical Sciences, 2010, 34(06):505-508.
[15] Cui C P, Wei P, Liu Y, et al. The protective role of Hepatopoietin Cn on liver injury induced by carbon tetrachloride in rats. Hepatol Res, 2009, 39(2):200-206.
[16] Liu Y, Saiyan S Y, Men T Y, et al. Hepatopoietin Cn reduces ethanol-induced hepatoxicity via sphingosine kinase 1 and sphingosine 1-phosphate receptors. J Pathol, 2013, 230(4):365-376.
[17] Chang J, Liu Y, Zhang D D, et al. Hepatopoietin Cn suppresses apoptosis of human hepatocellular carcinoma cells by up-regulating myeloid cell leukemia-1. World J Gastroenterol, 2010, 16(2):193-200.
[18] Wang S S, Wang Y L, Lu Q S, et al. The expression and distributions of ANP32A in the developing brain. Biomed Res Int, 2015, 2015:207347. |
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