Monday, August 13, 2012
Columbia Hall, Terrace Level (Washington Hilton)
Cutinase (EC 3.1.1.74) is a carboxylic ester hydrolases that is capable of degrading cutin polymers of plant cell wall. Cutinases can be considered as a link between esterases and lipases as they efficiently hydrolyze soluble esters and emulsified triacylglycerols and shows activity towards variety of soluble synthetic esters, insoluble triglycerides, synthetic fibers (polyethylene terephthalate fibers), and plastics (poly- caprolactone) etc. Escherichia coli have been extensively used for recombinant protein production for many years. The high level production in E. coli typically requires optimization of various physical and chemical parameters. In the present study, we optimized medium components and physical parameters for high level production of recombinant cutinase from Thermobifida fusca using pET expression system in E. coli BL21 (DE3). Six different media composition, including both defined and undefined media were screened with several other factors that are affecting the level of protein expression viz., concentration of IPTG, induction time, induction temperature and induction opportunity. The results showed that growth as well as recombinant cutinase production was considerably affected by the media composition. At optimized conditions, the production was almost 3-fold higher as compare to unoptimized conditions. Cellular localization study of the recombinant protein showed that most of the protein was localized in periplasm with no inclusion bodies. In conclusion, the study suggested that the employment of medium screening and optimization of various culture parameters as a part of recombinant protein production aid in improving the yield of recombinant proteins.