Expression and purification of an insect endoglucanase using an E. coli-based cell-free system

An E. coli-based cell-free extract was used to perform in vitro translation of an endogenous ?-1, 4-endoglucanase (Dvv-ENGase I) gene from the western corn rootworm beetle, Diabrotica virgifera virgifera, belonging to the glycoside hydrolase family (GHF) 45. The cDNA encoding Dvv-ENGase I was expressed as a 27-kDa polypeptide. Recombinant Dvv-ENGase I protein tagged with 6 poly Histidines was purified in one simple and single step via magnetocapture using Ni-based magnetic beads. The recombinant Dvv-ENGase I protein exhibited enzymatic activity not only on hydroxyethyl-cellulose (HEC), a nonionic and water-soluble cellulose polymer but also toward the fluorogenic 4-methylumbelliferyl-beta-D-glucopyranoside (4-MU-glucopyranoside). The applicability of E. coli-based cell-free expression system to the assembly of some insect genes indicates that it is possible obtain efficient and coupled transcription and translation of recombinant protein in a short period of time providing enough functionally active proteins for an array of downstream applications.