Honors Capstone Project
Date of Submission
Arts and Science
Capstone Prize Winner
Won Capstone Funding
Sciences and Engineering
Biochemistry | Biochemistry, Biophysics, and Structural Biology
Bovine (cow) rhodopsin is a 7‐transmembrane (7TM), lightabsorbing protein located in rod cells. It is activated by the photoisomerization of retinal, a Vitamin A derivative. This light‐triggered reaction activates the G‐Protein Coupled Receptor (GPCR), resulting in a signaling cascade within the cell. When previously cloned in an E. coli expression vector in the Braiman Lab, bovine rhodopsin expression was not successful, possibly due to E. coli’s lack of recognition of the foreign Nterminus portion of the protein, which may be a prerequisite for proper folding and insertion into the membrane. Our proposed solution is to create a chimera protein, replacing the N‐terminal leader sequence of bovine rhodopsin with proteorhodopsin’s N‐terminal leader sequence in order to successfully express this chimera in E. coli using the pBADTopo ® plasmid. Proteorhodopsin (pR), a protein found in marine bacterioplankton, with analogous photochemical activity to bovine rhodopsin, was chosen to serve as a candidate for creating this chimera due to its robust, successful protein expression, giving around 10mg/L of culture, expressed in the same plasmid and bacterial system. Using the restriction enzyme NcoI, we removed a portion of the gene encoding the N‐terminus of bovine rhodopsin, while PCR followed by NcoI digestion was used to amplify a similar‐sized portion of pR’s N‐terminus encoding DNA. These two DNA fragments were ligated, using T4 Ligase. Confirmation of successful ligation of these two DNA fragments to create the chimera protein‐encoding DNA is still pending.
Brown Jr., Chauncey, "Enhancing Bacterial Expression of Mammalian GProtein Coupled Receptors The Creation of a Proteorhodopsin‐Bovine Rhodopsin Chimera" (2009). Honors Capstone Projects - All. 461.
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