Expression and purification of recombinant human serpin B1 yields novel molecules with altered protease inhibitory activities: Functional implications

Abstract

Serpin B1 regulates the innate immune system by inhibiting serine and cysteine proteases that control programmed cell death and proliferation pathways. To provide recombinant human proteins for in vitro and in vivo studies we expressed and purified wild-type human serpin B1 and a C344A variant in the yeast S. cerevisiae. Both proteins expressed well and inhibited elastase and chymotrypsin. However, purification of wild-type serpin B1 in the absence of a reducing agent resulted in the specific loss of elastase - but not chymotrypsin - inhibition, concomitant with the formation of two higher molecular weight forms of the protein - a modified monomer and a dimer created via an intermolecular disulfide bond formed between C344 in respective serpin B1 monomers. In contrast to fully reduced serpin B1, both modified forms were good elastase substrates and catalytically cleaved at multiple adjacent sites within the reactive site loop. In contrast, purification of the C344A variant in the absence of a reducing agent yielded only one form of the protein which retained elastase and chymotrypsin inhibitory properties when purified. Furthermore, the elastase inhibitory activity of wild-type serpin B1, but not the C344A variant, was sensitive to oxidation. Thus, wild-type human serpin B1 should be formulated with a pharmaceutically acceptable reducing agent to protect C344 against post-translational oxidative modifications. Alternatively, the C344A variant of this protein may prove to be a suitable drug development candidate. These findings also suggest that inactivation of serpin B1 by oxidation may have a physiological role to play during inflammation.