National Center of Excellence in Molecular Biology (CEMB)
Abstract
Background: Enhanced production and improved properties of cellulases for a greater activity on plant biomass would rank amongst the top priorities for second-generation ethanol production. Based on the emergence of protein engineering as a cutting-edge technology for enhancing enzyme activity and expression level, the present study is aimed at the application of this technique to the major cellulosomal processing endoglucanase of C. thermocellum, CelR for refining enzyme characteristics. Methods: The full-length native enzyme gene (CelR) and a truncated version without the docking domains at C-terminus (CelR-CB) were PCR amplified using gene specific primers. The amplified PCR products were T/A cloned in the vector pTZ57 R/T and transformed in E. coli DH5α. The cellulase genes from the confirmed transformed plasmids were sub-cloned in T7 promoter-based expression vector pET-28a and expression analysis was done in E. coli (DE3) BL21 codon Plus. Results: An SDS PAGE analysis of both the CelR derivatives revealed that the truncated version i.e. CelR-CB showed a two-fold increase in expression level as compared to the full-length enzyme. Conclusion: The increased expression level of CelR in E. coli coupled with its increased production therefore makes it a promising method for augmenting the recombinant enzyme production for potential applications.
