2007 Spring Report
Michael TaShou Tang
The SBD group in our Lab work hard on several transformed E. Coli colonies for years. My assignment was about the proteins of one of them: the colony F58A (M.W. 11578.56 Dalton) which was specific site-direct mutated SBD inserted into vector pET-23a, the presentation, and the enzyme kinetic study. I got the colony (BL21-DE3) form Dr. Chou, and purified the SBD protein with ion-exchange column (purification data and SDS-PAGE data were not shown), and amylose resin column (BD). The result was that the amylose resin column might be a better way for purification of SBD protein.
After I got the purified SBD protein, the continuous processing steps were concentration and kinetic assay. The concentration process concludes the Amicon kits (10kD), and the final SBD concentration is about 0.7mg SBD/ml.
The enzyme kinetic analysis was according the theory which was developed by Skatchard. We use the scientific analytical software GrafPad Prism to find out the dissociated constant (Kd) and the maximum binding concentration (Bmax).
Here bellow is the kinetic data:
The goals here I shall proceed recently:
1. Prepare the new batches of transformed E. Coli., and store the batches with organized orders well.
2. Obtain large amount of the pure SBD-F58A, check the quality with SDS PAGE.