Purification, refolding of hybrid hIFNgamma-kringle 5 expressed in Escherichia coli.
The DNA sequence coding for plasminogen kringle 5 (pK5), an inhibitor of angiogenesis, was fused with that coding for interferon gamma and over-produced in the form of inactive inclusion bodies in E. coli. The amount of fusion protein was about 40% of total protein produced. The fusion protein contained in the inclusion bodies was solubilized in 8 M urea and purified by anion-exchange chromatography. We employed the orthogonal experimental design L16(4(5)) (5 factors, 4 levels, 16 experiments) procedure for researching the influence of denaturant, aggregation suppressor L-arginine, NaCl, pH, and glycine on the refolding procedure. Our results suggest that the presence of appropriate L-arginine, NaCl, and denaturant in the refolding buffer inhibits the aggregation of the fusion protein and increases the yield of renatured protein with biological activity. The refolded fusion protein, gammaIFN/pk5, has in vitro anti-endothelial cell proliferation activity.
Lu
H
H
Institute of Genetics, School of Life Sciences, Fudan University of Shanghai, People's Republic of China. honglu@shtel.net.cn
Zhang
H
H
Wang
Q
Q
Yuan
H
H
He
W
W
Zhao
Z
Z
Li
Y
Y
D020533:Angiogenesis Inhibitors / Q000494:pharmacology; D043168:Angiostatins; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004730:Endothelium, Vascular / Q000187:drug effects; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D002479:Inclusion Bodies / Q000378:metabolism; D007371:Interferon-gamma / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D018082:Kringles / Q000235:genetics*; D010446:Peptide Fragments / Q000627:therapeutic use; D010958:Plasminogen / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology / Q000627:therapeutic use; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D011994:Recombinant Proteins
D020533:Angiogenesis Inhibitors; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D007371:Interferon-gamma; D043168:Angiostatins; D010958:Plasminogen