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Title 

In vitro formation of active carboxypeptidase Y from pro-carboxypeptidase Y inclusion bodies by fed-batch operation

Authors 

Moon Sun HahmBong Hyun Chung

Publisher 

The Korean Society for Applied Microbiology

Issue Date 

2001

Citation 

Journal of Microbiology and Biotechnology, vol. 11, no. 5, pp. 887-889

Keywords 

carboxypeptidase Y (CPY)E. colipro-carboxypeptidase Y (pro-CPY)proteinase Krefoldingcarboxypeptidase Cenzyme precursorcell inclusioncontrolled studycytoplasm

Abstract 

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 μg/ml and 0.6 μg/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 1 ml of the renaturation buffer containing 60 μg/ml of proteinase K to give a final proteinase K concentration of 0.6 μg/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.

ISSN 

1017-7825

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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