Abstract
Tumstatin is a M(r) 28,000 C-terminal NC1 fragment of type alpha3 (IV) collagen that inhibits pathological angiogenesis and suppresses proliferation of endothelial cells and growth of tumors. We report here high cytoplasmic expression of recombinant human tumstatin in Escherichia coli and its purification, in vitro refolding, and inhibitory activity analysis. Human tumstatin was expressed in the bacterial cytoplasm as an insoluble N-terminal polyhistidine tagged protein, which accounted for more than 30% of total bacterial protein in BL21 (DE3) cells. After extraction and solubilization in guanidine-HCl, recombinant protein was purified to homogeneity using a simple one-step Ni(2+)-chelate affinity chromatography and then refolded by dialysis against acidic pH buffers with gradually decreasing concentrations of denaturant. The renatured recombinant tumstatin could specifically inhibit endothelial cell proliferation in a dose-dependent manner, and suppress bFGF-induced angiogenesis in chick embryo chorioallantoic membrane and tumor growth in mouse B16 melanoma xenograft models.
References
Sep 1, 1991·Bio/technology·T KiefhaberJ Buchner
Jul 1, 1992·European Journal of Pediatrics·L Schweigerer, T Fotsis
Jan 1, 1995·Nature Medicine·J Folkman
Aug 9, 1996·Cell·D Hanahan, J Folkman
Oct 3, 1999·Methods in Enzymology·E De Bernardez ClarkR Rudolph
Jan 8, 2000·The Journal of Biological Chemistry·G D KamphausR Kalluri
Mar 14, 2000·The Journal of Biological Chemistry·E PetitclercP C Brooks
Apr 15, 2000·The Journal of Biological Chemistry·Y MaeshimaR Kalluri
Feb 7, 2001·Proceedings of the National Academy of Sciences of the United States of America·M RehnK Vuori
Mar 30, 2001·The Journal of Biological Chemistry·Y MaeshimaR Kalluri
Oct 12, 2002·Journal of Cell Science·Nathalie Ortega, Zena Werb
Jul 29, 2003·Biotechnology Letters·Hee Kyoung JeonIn Sik Chung
Dec 19, 2003·Biochemical and Biophysical Research Communications·Guo-An HeRui-Fang Li
Mar 25, 2004·Protein Expression and Purification·Kyung Hwa ChangIn Sik Chung
Jun 28, 2005·Biochemical and Biophysical Research Communications·Yuki Hamano, Raghu Kalluri
Nov 13, 2008·Cytotechnology·Jong-Hwa ParkIn Sik Chung
Citations
Nov 13, 2013·Protein Expression and Purification·Venugopal GundaYakkanti A Sudhakar
Mar 14, 2013·PloS One·Alen FaizJanette K Burgess
Mar 5, 2013·Biochemical and Biophysical Research Communications·Jessica ThevenardSylvie Brassart-Pasco
Jul 26, 2013·Spermatogenesis·Elissa W P Wong, C Yan Cheng
Jun 20, 2014·Anti-cancer Drugs·Quliang GuLijing Wang
Aug 19, 2009·Molecular Biology Reports·Chun-xiao XuJia-xuan Xin
May 15, 2015·Technology in Cancer Research & Treatment·C WeiW S Zeng
Jul 18, 2006·Journal of Biochemistry·Jieru MengYingqi Zhang