Overexpression and protein folding of a chimeric β<span style="font-size:16.0pt;font-family:HiddenHorzOCR;mso-bidi-font-family:HiddenHorzOCR">-glucosidase constructed from <i>Agrobacterium tumefaciens </i>and <i>Cellvibrio gilvus</i> </span>

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

<i>Agrobacterium&nbsp; tumefaciens </i>with the corresponding homologous region of <i>Cellvibrio gilvus </i><span style="mso-bidi-font-style:italic">β-glucosidase. The chimeric

gene was overexpressed in <i>E. coli </i>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, <i>in vivo. </i>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 <i>in vitro </i>conditions using various dialysis strategies. Dialysis.

rapid dilution and a newly devised method of folding immobilized proteins yielded active enzyme. The usefulness of the <i>in</i>

<i>vitro </i>folding methods to obtain functional enzymes from overproduced but non-functional proteins has been discussed.

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