Engineering, Expression and Renaturation of Targeted TGF-Beta Fusion Proteins

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Abstract
This study reports the expression, purification, and renaturation of biologically active Transforming Growth Factor-beta1 (TGF-β1) fusion proteins from Escherichia coli (E. coli). A prokaryotic expression vector was engineered to produce tripartite fusion proteins consisting of (i) a purification tag, (ii) a protease-sensitive linker/collagen binding domain, and (iii) a cDNA sequence encoding the active fragment of human TGF-β1. The expressed fusion proteins TGF-B1-F1 and TGF-B1-F2, located in inclusion bodies, were solubilizcd with 8 M urea and renatured using a glutathione redox-coupled system and protracted dialysis under several experimental conditions. The purification of the recombinant proteins was achieved by binding the His-tag of the fusion proteins on a Ni-NTA metal chelate column. The biological activity of the recombinant growth factor was demonstrated by its ability to inhibit mink lung (MvlLu) cell proliferation and/or to stimulate proliferation of NIH-3T3 mouse fibroblasts, where purified human platelet TGF-β1 served as a positive control. Purified TGF-B1-F1 and TGF-B1-F2 (collagen binding) constructs exhibited anti-proliferative activities comparable to purified platelet TGF-β1, but at lower specific activities. Binding of the renatured TGF-B1-F2 fusion protein to collagen was demonstrated by stable binding on a collagen-conjugated Sephadex-G 15 column. The high affinity binding was also demonstrated by the binding of 3H-collagen to the TGF-B1-F2 protein immobilized on a Ni-NTA column. The TGF-B1-F2 fusion protein bound to collagen coated surfaces with high affinity but exhibited comparatively lower biological activity than the fusion protein in solution, suggesting a potentially latent configuration. Taken together, these results demonstrate that biologically active TGF-β1 fusion proteins can be recovered from transformed bacteria by oxidative refolding; thus, providing a means for its high-yield production, purification, and renaturation from microorganisms. Furthermore, these results support the concept that auxiliary domains may be used to modulate and/or target TGF-β1 for specific applications.