Segment Cloning and Expression of Human VIGILIN Gene

YU Xiao-qin, WEI Ling, LIU Qiu-ying. et al

Abstract

To investigate the mechanisms of interaction between high-density lipoprotein binding protein (HDLBP)-VIGILIN with other proteins, we cloned VIGILIN cDNA N, KH1-7, KH8-12, KH13-14, and C fragments separately into expression vector, and identify the expressed proteins. Methods The recombinant plasmid pDsred2-N1/ VIGILIN was used as template to amplify VIGILIN full length, VIGILIN Nterminal, KH1-7, KH8-12, KH13-14, C terminal and recombinated them with pGEX 5X 3. After transformed into E.coli BL21 cells, the recombinants were confirmed by enzyme digestion and sequence analysis. After optimizing the IPTG inducing condition, we induced GST-VIGILIN fusion proteins on the appropriate conditions. Results The recombinant plasmids of pGEX 5X 3/ VIGILIN FL, pGEX 5X 3/ VIGILIN N terminal, pGEX 5X 3/ VIGILIN KH1-7, pGEX 5X 3/ VIGILIN KH8-12, pGEX 5X 3/ VIGILIN KH13-14, pGEX 5X 3/ VIGILIN C terminal were constructed successfully, and induced the GST-VIGILIN fusion proteins. Conclusion pGEX 5X 3/ VIGILIN FL, pGEX 5X 3/ VIGILIN N terminal, pGEX 5X 3/ VIGILIN KH1-7, pGEX 5X 3/ VIGILIN KH8-12, pGEX 5X 3/ VIGILIN KH13-14, pGEX 5X 3/ VIGILIN C terminal recombinant plasmids were constructed successfully, and their corresponding fusion proteins were successfully expressed.

 

Keywords: VIGILIN, Domainб Segmented cloning, Prokaryotic expression

 

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References


Wen WL, Stevenson AL, Wang CY, eial. Vgll, a multi-KH domain protein, is a novel component of the fission yeast stress granules required for cell survival under thermal stress. Nucleic Acids Res, 2010; 38( 19): 6555-6566.

Batlle M. Marsellach FX, Huertas D, et al. Drosophila vigilin. DDP1, localises to the cytoplasm and associates to the rough endoplasmic reticulum. Biochim Biophys Acta, 2011; 1809 ( 1 ); 46-55.

Rohwedel J, Kiigler S, Engebrecht T, et al. Evidence for posttranscriptional regulation of the multi К homology domain protein VIGILIN by a small peptide encoded in the 5, leader sequence. Cell Mol Life Sci, 2003; 60(8); 1705-1715.

Young SG, Davies BS. Fong LG, et al. GPIHBP1; an endothelial cell molecule important for the lipolytic processing of chylomicrons. Curr Opin Lipidol, 2007; 18(4);389-396.

Huertas D , Cortes A , Casanova J, et al . Drosophila DDP1, a multi KH domain protein contributes to centromeric silencing and chromosome segregation. Curr Biol, 2004; 14 ( 18) ; 1611- 1620.

Li AM, Watson A, Fridovich-Keil JL. Scpl60p associates with specific mRNAs in yeast. Nucleic Acids Res, 2003; 31 ( 7 ) : 1830-1837.

Liu QY, Yang B, Xie XY-et al. Vigilin interacts with CTCF and involves in CTCF-dependent regulation of imprinted genes Igf2/H19. FEBS J ,2014;281 (12):2713-2725.

Yang WL, Wei L, Huang WQ, et al. Vigilin is overexpressed in hepatocellular carcinoma and is required for HCC cell proliferation and tumor growth. Oncol Rep, 2011; 31 (5); 2328- 2334.

Woo HH, Yi X. Lamb T, et al. Posttranscriptional suppression of proto-oncogene c-fms expression by vigilin in breast cancer. Mol Cell Biol,2011;31 (1);215-225.

Young SG, Davies BS, Voss CV, et al. GPIHBP1, an endothelial cell transporter for lipoprotein lipase. J Lipid Res, 2011;52(11):1869-1884.

Lu SH, Jeon AH. Schmitt-Ulms G, et al. Vigilin interacts with signal peptide peptidase. Proteome Sci,2012; 10 ( 1); 10- 33.

Hirschmann WD, Westendorf H, Mayer A, et al. Scpl60p is required for translational efficiency of codon-optimized mRNAs in yeast. Nucleic Acids Res,2014 ;42(6) :4043-4055.

Zhou J, Wang QQ, Chen LL, et al. On the mechanism of induction of heterochromatin by the RNA-binding protein vigilin. RNA,2008; 14(9): 1773-1781.

Shen WY, Liu QY, Wei L, et al. CTCF-mediated reduction of vigilin binding affects the binding of HPla to the satellite 2 locus. FEBS Lett,2014 ;588(9): 1549-1555.


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