Date of Completion
Biochemical and Biomolecular Engineering | Chemical Engineering | Molecular Biology | Molecular, Cellular, and Tissue Engineering
This report details the results of an ongoing project to engineer a mutant form of Red Fluorescent Protein (RFP) variant mCherry that acts as a real-time in vivo protease sensor. The sought-after mutant only becomes fluorescent when exposed to Tobacco Etch Virus (TEV) Protease, this system’s model protease. This will be accomplished via the insertion of the TEV Protease Recognition Site (TEV-PRS) in such a position that, before cleavage, will prevent the protein from folding to fluorescent conformation, but upon cleavage, will allow for fluorescent conformation to occur. The cylindrical structure of the protein, composed of beta-pleated sheets, contains “loops” connecting these sheets, was analyzed, and sixteen sites were identified within these loops as candidates for insertion sites of the TEV-PRS. Using Site-Directed Mutagenesis (SDM) and Overlap-Extension Polymerase Chain-Reaction (OE-PCR), insertion at twelve of the identified sites has been attempted, two of which have been expressed in surface display yeast, digitally imaged, and their initial (pre-cleavage) fluorescence has been quantified. Four of the intended sites were successfully mutated, in addition to one accidental site. While the sought after mutant has not yet been identified, the results at this stage indicate promise, and further study is recommended and will be carried on in the future by other Research Assistants.
Kinard, Thomas C., "Engineering a Fluorescent Protease Sensor for in vivo Protein Detection" (2017). Honors Scholar Theses. 591.