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Title: Overexpression and protein folding of a chimeric β-glucosidase constructed from Agrobacterium tumefaciens and Cellvibrio gilvus
Authors: Singh, S P
Kim, J D
Machida, S
Hayashi, K
Issue Date: Aug-2002
Publisher: NISCAIR, CSIR
Abstract: In continuation of our investigation on structure and function relationship of β-glucosidases from mesophilic and thermophilic bacteria, we constructed a chimeric gene by shuffling 17% length in C terminal region of β-glucosidase of Agrobacterium  tumefaciens with the corresponding homologous region of Cellvibrio gilvus β-glucosidase. The chimeric gene was overexpressed in E. coli BL21 (DE3) using pET vector. However, nearly all of the β-glucosidase produced was trapped into inclusion bodies in catalytically non-functional state. Attempts were made to solubilize the overexpressed protein by co-expression with molecular chaperone, GroEL/ES, in vivo. The molecular chaperone assisted protein folding that had earlier yielded encouraging results, did not improve the solubilization in the present case with a chimeric β- glucosidase. Further, we explored protein renaturation under in vitro conditions using various dialysis strategies. Dialysis. rapid dilution and a newly devised method of folding immobilized proteins yielded active enzyme. The usefulness of the in vitro folding methods to obtain functional enzymes from overproduced but non-functional proteins has been discussed.
Page(s): 235-239
ISSN: 0975-0959 (Online); 0301-1208 (Print)
Appears in Collections:IJBB Vol.39(4) [August 2002]

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