Soluble Expression of Recombinant TGF-β in Bacteria

Transforming growth factor beta (TGF-β) isoforms are 25-kDa homodimeric polypeptides that play crucial non-overlapping roles in embryogenesis, tissue development, carcinogenesis and immune regulation. The monomers have nine cysteines, eight of which form four internal disulfides and one of which participates in the formation of the inter-chain disulfide. TGF-βs and other proteins of the superfamily are expressed as much larger latent proteins (390 amino acids for TGFβ1 and 412 amino acids for TGFβ2 and TGFβ3), which contain a signal region, a pro-region and a C-terminal region, all three of which are cleaved by a variety of mechanisms (e.g., proteolysis, alkaline lysis) to yield the active protein by 112 amino acids. The approaches used to produce the proteins of the superfamily include: 1) overexpression of the full-length pre-pro proteins in cultured eukaryotic cells, and 2) overexpression of the mature monomers (residues 279-390) in bacteria as inclusion body, followed by renaturation of the overexpressed polypeptide into native dimers. So, production of correctly mature TGF-βs in dimer form, remains a challenge. SHuffle is a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. In this study, we expressed recombinant antagonistic form of TGF-β in SHuffle bacteria. Firstly, pET28a containing TGF-β gene was transformed into SHuffle competent cells. Then three parameters, including temperature of growth and expression, time and strength of induction, were optimized. Expressed recombinant TGF-β antagonist was purified by Ni-NTA agarose and its molecular weight determined on SDS-PAGE under reducing conditions and non- reducing conditions. Mink cell proliferation assay showed that this dimeric antagonistic TGF-β was bioactive.