In Vitro Formation of Active Carboxypeptidase Y from Pro-Carboxypeptidase Y Inclusion Bodies by Fed-Batch Operation

In Vitro Formation of Active Carboxypeptidase Y from Pro-Carboxypeptidase Y Inclusion Bodies by Fed-Batch Operation

The gene encoding yeast pro-carboxypeptidase Y (pro-CPY) has been cloned and expressed in Escherichia coli. Most of the expressed pro-CPY was accumulated as cytoplasmic insoluble aggregates. In our previous study, active CPY was obtained by renaturation of entirely denatured pro-CPY followed by in vitro proteolytic processing with proteinase K along with the activation process. The same refolding process was performed to produce an active CPY from pro-CPY inclusion bodies with renaturation buffers containing proteinase K at different concentrations. The refolding efficiency decreased from $25\%\;to\;2\%$ in the renaturation buffers containing proteinase K at concentrations of $60{\mu}g/ml\;and\;0.6{\mu}g/mi$, respectively. In an attempt to increase the refolding efficiency with a lesser amount of proteinase K, a novel fed-batch refolding process was developed. In a fed-batch refolding, 99 ml of the renaturation buffer containing pro-CPY was gradually added into 1 ml of the renaturation buffer containing $60{\mu}g/ml$ of proteinase K to give a final proteinase K concentration of $0.6{\mu}g/ml$. The fed-batch refolding process resulted in a refolding efficiency of $18\%$, which corresponded to a 9-fold increase over that ($2\%$) in the batch process.