REFOLDING OF A RECOMBINANT COLLAGEN-TARGETED TGF-BETA-2 FUSION PROTEIN EXPRESSED IN ESCHERICHIA-COLI

In this study, a tripartite transforming growth factor-beta (TGF-beta 2) fusion protein bearing an N-terminal purification tag and an auxili ary collagen binding decapeptide has been constructed and expressed at high levels in Escherichia coli. The resulting recombinant protein ac cumulates in an insoluble and biologically inactive inclusion-body com plex. The insoluble protein was solubilized in guanidine hydrochloride and a Ni-chelating affinity column was utilized to isolate the 13.5-k Da TGF-beta 2 fusion protein, which was then refolded into its native conformation under controlled redox conditions. The formation of nativ e homodimers was monitored by nonreducing sodium dodecyl sulfate-polya crylamide gel electrophoresis gradient gels and the bioactivity determ ined by a quantitative TGF-beta assay system using mink lung epithelia l cells transfected with a plasminogen activator inhibitor-1 promoter/ luciferase reporter plasmid. To optimize yields, renaturation conditio ns including denaturants, limiting protein concentrations, redox ratio s, dialysis conditions, and refolding kinetics were studied and monito red by bioactivity. These studies demonstrate that recombinant TGF-bet a 2 fusion proteins can be produced in E. coli and renatured into biol ogically active homodimers. Furthermore, they confirm that the auxilia ry collagen binding domain effectively targets the recombinant growth factor to type I collagen. Taken together, these studies advance the t echnology necessary to generate large quantities of targeted TGF-beta fusion proteins for specific biomedical applications. (C) 1997 Academi c Press.