Overexpression and protein folding of a chimeric beta-glucosidase constructed from Agrobacterium tumefaciens and Cellvibrio gilvus.
In continuation of our investigation on structure and function relationship of beta-glucosidases from mesophilic and thermophilic bacteria, we constructed a chimeric gene by shuffling 17% length in C terminal region of beta-glucosidase of Agrobacterium tumefaciens with the corresponding homologous region of Cellvibrio gilvus beta-glucosidase. The chimeric gene was overexpressed in E. coli BL21 (DE3) using pET vector. However, nearly all of the beta-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 beta-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.
Enzyme Application Laboratory, National Food Research Institute, Tsukuba, 305 Japan 2Department of Biosciences, Saurashtra University, Rajkot, Gujarat 360 005, India. satyapsingh@yahoo.com
S P
SP
Singh
J D
JD
Kim
S
S
Machida
K
K
Hayashi
D016960:Agrobacterium tumefaciens / Q000201:enzymology*; D020633:Cellvibrio / Q000201:enzymology*; D018835:Chaperonin 10 / Q000737:chemistry; D018834:Chaperonin 60 / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D001467:Hordeum / Q000201:enzymology; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013696:Temperature; D001617:beta-Glucosidase / Q000096:biosynthesis / Q000737:chemistry*
D018835:Chaperonin 10; D018834:Chaperonin 60; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; D001617:beta-Glucosidase