Determining the Enzymatic Function of a SaCD00432683 Vector-Derived Protein

Abstract

Protein characterization is a fundamental aspect of biochemical research, including but not limited to the study of genetic expression, biochemical processes, and medicine development. The protein that is expressed by the SaCD00432683 vector insert sequence has a known structure, but the enzymatic function is yet to be known. Here, we seek to characterize this protein expressed from SaCD00432683 to determine its enzymatic function through vector transformation and protein expression, purification techniques including affinity chromatography and SDS-PAGE, database exploration, and DNA sequencing.  The agarose gel electrophoresis showed that the purified PCR product, which corresponds to the ampicillin-resistant gene, had a band at ~1000 bp. The SDS-PAGE results showed that the protein of interest had a band at ~70 kDa, as expected. The in-silico research using the Protein Data Bank, BLAST, and Interpro helped us predict that our protein is in the alpha/beta hydrolase superfamily. The two relevant domains are alpha/beta hydrolase and BD-FAE-like domain, which cleave ester bonds. This helped us predict our protein has a hydrolytic function. We were able to successfully grow and purify our protein which will be used in our next steps, which are to conduct enzyme kinetic and activity assays to confirm that our protein has hydrolytic activity