First Name:
Shea
Last Name:
Comadoll
Major Department:
Chemistry
Thesis Director:
Jennifer Cecile
Date of Thesis:
May 2013
Transmembrane proteins are essential to many biological processes yet appear in only 2% of known crystal structures. Due to limitations with effective protein expression and solubility, structural and functional information on transmembrane proteins lags behind what is known about soluble proteins. One class of these transmembrane proteins is the organic anion transporters (OATs). OATs are responsible for the transport of negatively charged drugs, xenobiotics, and metabolites in the body (specifically in epithelial tissues). Biosynthesizing this protein in bacteria (Escherichia coli, E. coli) would enable more functional in vitro experiments for pharmaceutical targeting, as well as provide protein for functional studies. Within this work subcloning protocols were employed to insert the human organic anion transporter isoform 1 (hOAT1) into a traditional E. coliexpression vector (pET-19b). Once complete the pET-19b vector containing hOAT1 was then transformed into BL21 (DE3) E. coli cells. Screening for presence of the pET-19b vector and hOAT1 was done using LB agar plates containing ampicillin and colony PCR. All colonies growing on the plates had confirmation of a transformation, however, presence of the hOAT1 sequence cannot be confirmed until further testing has been done. Cells grown on plated were induced to get protein expression. Post induction, cells were stored in glycerol solutions.