Lopez, Harry , Gillikin, Jeff , Holmes, Robert , Boston, Rebecca .
Production of Recombinant Maize Ubiquiting Conjugating Enzyme in E.coli.
THE endoplasmic reticulum (ER) is a network of membranous tubules in the cytoplasm of a cell which is equipped with molecular chaperones and enzymes essential to protein folding. Correctly folded proteins are exported from the endoplasmic reticulum, while mis-folded proteins are retained and selectively degraded by a biological process known as ER-associated degradation (ERAD). Ubiquitin conjugating enzyme (Ubc1) mediates selective degradation of short-lived and abnormal proteins, and is predicted to be important in ERAD. Our goal was to produce recombinant Ubc1 in an IPTG-inducible E. coli expression system so that it could be used for antibody production and protein interaction studies. We used a pRSET plasmid vector that would produce a fusion protein with polyhistidine and Xpress® epitope tags along with the maize Ubc1 coding sequence. Optimal protein production was achieved by testing different growth temperatures, IPTG concentrations, and growth phase of the E. coli culture at induction. Protein solubility was analyzed by centrifugation following different buffer treatments. To detect the recombinant Ubc1, we used a combination of western blotting and staining of SDS-polyacrylamide gels with Coomassie Brilliant Blue. Large-scale production and purification of Ubc1 was carried out based on the optimal production parameters. Based on protein yields we conclude that maize Ubc1 can be produced as a soluble protein in E. coli and effectively purified by affinity chromatography.
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1 - University of Puerto Rico at Cayey, Deparment of Biology, Box 664, Naranjito, P.R, 00719, Puerto Rico
2 - North Carolina State University, Department of Botany, Campus Box 7612, Raleigh, North Carolina, 27695-7612, USA
Maize, ER, Ubiquitin, plasmid vector.
Presentation Type: Poster:Posters for Sections
Location: Auditorium/Bell Memorial Union
Date: Tuesday, August 1st, 2006
Time: 12:30 PM