Cloning And Expression Of Cel5a Gene From Acidothermus cellulolyticus In E.coli BL21 Codon Plus

Abstract

The exploitation of plant biomass due to its inexhaustible nature is an imperative need for the production of cellulases which show a high specific activity against cellulose if the potential agriculture and industrial benefits of plant biomass are to be fully realized. An acidothermophilic cellulose degrading organism called Acidothermus cellulolyticus strain 11B produces multiple acidic thermophilic cellulases. Out of all the cellulase producing genes, Cel5A (EI beta-1,4- endoglucanase) was amplified by PCR using the genomic DNA as a template. The amplified fragment was ligated in T-A cloning vector using the tailing technique. The resultant plasmid Cel5A/pTZ57R/T was transformed into E. coli DH5α cells. Positive colonies were screened for the presence of insert. The excised and purified gene fragments were then ligated in NdeI/XhoI double digested pET22b(+) vector. The recombinant construct was transformed into E. coli BL21 CodonPlus RIPL, under the control of IPTG-inducible T7-lac promoter. SDS-PAGE analysis showed that a protein of molecular weight 34kDa was expressed in the insoluble fraction of the host bacterium. An expression level of approximately 28% of the total cell protein was obtained with a minimal activity of 0.275 U. A mixture of genetically modified plant biomass and promising cellulases whether identified from natural sources or synthetically enhanced may be required in combination for the final critical breakthrough in this area of research.