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KMID : 0545120010110050887
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Journal of Microbiology and Biotechnology
2001 Volume.11 No. 5 p.887 ~ p.889
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In Vitro Formation of Active Carboxypeptidase Y from Pro-Carboxypeptidase Y Inclusion Bodies by Fed-Batch Operation
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HAHM MOON-SUN
CHUNG BONG-HYUN
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Abstract
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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 [3], 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㎍/ml and 0.6㎍/ml, 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 I ml of the renaturation buffer containing 60 ㎍/ml of proteinase K to give a final proteinase K concentration of 0.6 ㎍/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.
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