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.