Oxidative refolding of recombinant prochymosin

Abstract

The disulphide-coupled refolding of recombinant prochymosin from Escherichia coli inclusion bodies was investigated. Prochymosin solubilized from inclusion bodies is endowed with free thiol groups and disulphide bonds. This partially reduced form undergoes renaturation more efficiently than the fully reduced form, suggesting that some native structural elements existing in inclusion bodies and remaining after denaturation function as nuclei to initiate correct refolding. This assumption is supported by the finding that in the solubilized prochymosin molecule the cysteine residues located in the N-terminal domain of the protein are not incorrectly paired with the other cysteines in the C-terminal domain. Addition of GSH/GSSG into the refolding system facilitates disulphide rearrangement and thus enhances renaturation, especially for the fully reduced prochymosin. Based on the results described in this and previous papers [Tang, Zhang and Yang (1994) Biochem. J. 301, 17-20], a model to depict the refolding process of prochymosin is proposed. Briefly, the refolding process of prochymosin consists of two stages: the formation and rearrangement of disulphide bonds occurs at the first stage in a pH11 buffer, whereas the formation and adjustment of tertiary structure leading to the native conformation takes place at the second stage at pH8. The pH11 conditions help polypeptides to refold in such a way as to favour the formation of native disulphide bonds. Disulphide rearrangement, the rate-limiting step during refolding, can be achieved by thiol/disulphide exchange initiated by free thiol groups present in the prochymosin polypeptide, GSH/GSSG or protein disulphide isomerase.