Modification, Verification of Sequence and Optimization of Expression of P297F an Inactive Mutant of Flavonol Specific Glucosyltransferase from Grapefruit (CP3GT)

• Main Author: Fox, Sarah
• Format: Others
• Published: Digital Commons @ East Tennessee State University 2020
• Subjects: structure/function; glucosyltransferase; flavonol; flavonoids; Biochemistry; Plant Biology
• Online Access: https://dc.etsu.edu/honors/557; https://dc.etsu.edu/cgi/viewcontent.cgi?article=1661&context=honors

Citrus fruits are widely consumed and can offer various health benefits. One enzyme found in grapefruits, Citrus paradisi flavonol specific 3-O-glucosyltransferase (CP3GT), catalyzes the addition of glucose to one specific flavonoid class and at only one site. These flavonoids are plant secondary metabolites that can be used in a variety of plant functions including signaling and protection. The only class of flavonoids that CP3GT glucosylates is flavonols, and this specificity is of interest to study for potential benefits in biotechnology and enzyme modeling. In order to study this enzyme and its structure, a variety of mutants were created using site-directed mutagenesis. One mutant, P297F, exhibited a loss of function. This mutant was previously studied by inserting a thrombin cleavage site, extracting the plasmid expressing the mutation and sequencing it. The gene sequence was previously verified to be in frame and contain the needed thrombin cleavage site to remove tags used for protein purification and identification. The plasmid was then linearized, and transformed into yeast. After this, conditions for protein expression were tested over a 72-hour period. The protein was found to have optimal expression at 50 hours with a constant temperature of 28 °C and methanol concentration of 0.5 %. However, numerous protein expression experiments indicated very low protein expression. For this reason, the P297F gene was amplified through colony PCR, extracted and sent for sequencing to verify the transformation of the gene into yeast and identify possible reasons for low protein production. Analysis of this sequencing data showed a single nucleotide addition early in the tag sequence causing a frameshift after this location. Reanalysis of the previous plasmid sequencing data showed this same mutation, indicating improper conclusions were drawn. Efforts should be made to identify a plasmid without the mutation or correct the frameshift mutation so that the tag sequence produces the correct amino acids.