Peptide disulfides CGC and RKCGC facilitate oxidative protein refolding

Oxidative protein refolding is a big challenge for the recovery of disulfide bonds containing recombinant proteins expressed as inclusion bodies. In this study, the disulfide form of a small peptide mimic of protein disulfide isomerase, CGC, was explored to facilitate oxidative protein refolding with residual dithiothreitol (DTT) as the reductant. Working with DTT, CGC gave higher yield and folding rate than oxidized glutathione. Then, a new pentapeptide, RKCGC disulfide, was designed. It was found that RKCGC was more effective than CGC in increasing the folding rate and final yield, as demonstrated in the oxidative refolding of lysozyme and ribonuclease A. It was because the pentapeptide has lower pKa and higher reduction potential than CGC. Moreover, it efficiently facilitated protein refolding not only at neutral pH but also in a weak alkaline buffer. With CGC or RKCGC as the oxidant, the DTT removal step and the addition of other thiol reductants were not needed. This would benefit in decreasing the processing time and cost in protein refolding processes. This design has thus provided more efficient oxidant, and it may pave the way for the design of more effective thiol-disulfide redox agents.

► A new oxidant, RKCGC disulfide, was designed to catalyze oxidative protein refolding. ► RKCGC facilitated oxidative protein refolding more effectively than CGC disulfide. ► RKCGC was effective not only in neutral pH but also in weak alkaline solutions. ► The design reveals that adding nearby positive charges to peptide disulfide is effective to improve its catalytic activity by lowering the pKa of the corresponding thiol.