Purification and characterization of RGD tumor-homing peptide conjugated human tumor necrosis factor alpha over-expressed in Escherichia coli.
A number of approaches have been investigated to enhance the selective toxicity of tumor necrosis factor alpha (TNFalpha) to permit its systemic use in cancer therapy. Because vascular targeting has been proven to be a valid strategy for improving the therapeutic index of TNFalpha, we prepared RGD-hTNF consisting of human TNF fused with the ACDCRGDCFCG peptide, a ligand of alpha(v)beta(3) and alpha(v)beta(5) integrins. Recombinant RGD-hTNF was produced in Escherichia coli as a polyhistidine fusion protein. Between polyhistidine tag and RGD-hTNF, a tobacco etch virus (TEV) protease cleavage site (ENLYFQG) was introduced to ensure the release of intact RGD-hTNF. The purification strategy consisted of the target protein capture step by immobilized metal affinity chromatography (IMAC), TEV protease cleavage of fusion protein, the subtractive depletion of removed His-tag by IMAC and the final gel filtration step. As a result, about 18 mg of intact RGD-hTNF was obtained from 1l of bacteria culture. The purified RGD-hTNF was characterized by SDS-PAGE, Western blot, mass spectroscopy and gel filtration. Since the RGD-hTNF molecule retained the cytotoxic activity of the TNF moiety and the integrin binding ability of the RGD moiety, the purification method provided material for assessing its anti-tumor activity in animal model.
Ma
Dingyuan
D
The State Key Laboratory of Pharmaceutical Biotechnology, College of Life Sciences, Nanjing University, Nanjing 210093, PR China.
Chen
Yuan
Y
Fang
Lei
L
Jin
Guanghui
G
Zhou
Bin
B
Cao
Lin
L
Ye
Jianqiang
J
Hua
Zichun
Z
D000818:Animals; D002448:Cell Adhesion; D002846:Chromatography, Affinity; D010450:Endopeptidases / Q000378:metabolism; D004765:Enteropeptidase / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D015870:Gene Expression; D006639:Histidine / Q000378:metabolism; D006801:Humans; D051379:Mice; D009369:Neoplasms / Q000628:therapy*; D009842:Oligopeptides / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D021241:Spectrometry, Mass, Electrospray Ionization; D014409:Tumor Necrosis Factor-alpha / Q000302:isolation & purification* / Q000378:metabolism
C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; C439524:TNF protein, human; D014409:Tumor Necrosis Factor-alpha; D006639:Histidine; C047981:arginyl-glycyl-aspartic acid; D010450:Endopeptidases; C099488:TEV protease; D004765:Enteropeptidase