Abstract |
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.
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Authors | Dingyuan Ma, Yuan Chen, Lei Fang, Guanghui Jin, Bin Zhou, Lin Cao, Jianqiang Ye, Zichun Hua |
Journal | Journal of chromatography. B, Analytical technologies in the biomedical and life sciences
(J Chromatogr B Analyt Technol Biomed Life Sci)
Vol. 857
Issue 2
Pg. 231-9
(Oct 01 2007)
ISSN: 1570-0232 [Print] Netherlands |
PMID | 17716959
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
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Chemical References |
- His-His-His-His-His-His
- Oligopeptides
- Recombinant Fusion Proteins
- TNF protein, human
- Tumor Necrosis Factor-alpha
- Histidine
- arginyl-glycyl-aspartic acid
- Endopeptidases
- TEV protease
- Enteropeptidase
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Topics |
- Animals
- Cell Adhesion
- Chromatography, Affinity
- Endopeptidases
(metabolism)
- Enteropeptidase
(metabolism)
- Escherichia coli
(metabolism)
- Gene Expression
- Histidine
(metabolism)
- Humans
- Mice
- Neoplasms
(therapy)
- Oligopeptides
(isolation & purification, metabolism)
- Recombinant Fusion Proteins
(isolation & purification, metabolism)
- Solubility
- Spectrometry, Mass, Electrospray Ionization
- Tumor Necrosis Factor-alpha
(isolation & purification, metabolism)
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