A bacterial expression system for the β-subunit of hCG (hCGβ) has been developed to produce suitable amounts of this protein for structural and biological studies. To produce hCGβ in Escherichia coli, the nucleotide sequence that encodes the amino acid leader sequence was removed from the hCGβ complementary DNA, and the gene was cloned into a pET expression vector. After induction of protein synthesis in host bacteria, recombinant hCGβ (rhCGβ) accumulated in inclusion bodies in an unfolded state. The inclusion bodies were purified from induced cultures of E. coli, solubilized in urea, and fractionated by reverse phase HPLC. In this way, 6-7 mg unfolded hCGβ were recovered from 1 liter culture. rhCGβ was folded in the presence of 6.4 mM cysteamine and 3.6 mM cystamine at pH 8.7 at a final concentration of 0.02 mg/ml protein. The folded protein assembled with urinary hCGα and the purified rhCGβ/urinary α dimer bound to and activated the human LH/CG receptor permanently expressed in a cell line, indicating that it was a functional hormone. The rhCGβ/urinary α dimer also stimulated in vivo ovulation in rats, thus confirming the biological activity of bacterially expressed hCGβ. Because E. coli lacks the ability to glycosylate proteins, these activity results indicate that the N-linked and O-linked oligosaccharides of hCGβ are not required for protein folding, subunit assembly, or full biological activity. The success of producing hCGβ in bacteria and of folding it in vitro implies that the β-subunits of the other members of the glycoprotein hormone family, LH, FSH, and TSH, can also be produced in this manner. This may facilitate structural studies of these hormones as well as lead to the production of recombinant hormones for biological studies and clinical use.
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