GENERATION OF A CYSTEINE-LESS BACKBONE YCF1 PROTEIN FOR ELECTRON PARAMAGNETIC RESONANCE SPECTROSCOPY

Abstract

There are a lot of research applications to YCF1 including research treatments for various diseases. One of those illnesses, cystic fibrosis, has a protein that does not work well known as the cystic fibrosis transmembrane conductance regulator, CFTR. YCF1 and CFTR are homologs, thus the research conducted on understanding cysteine modification and conformation space could be utilized for understanding how to improve current treatment options for cystic fibrosis. The goal of the research is to have a multiple cysteine-less mutated YCF1’s that are purified and ready for their confirmational space to be examined. The following was utilized for purification: primer design for mutagenesis, Q5 mini-prep, QIAprep spin miniprep kit, yeast growth, and nickel purification. The primary technique being used to examine the conformational space is EPR, and Cryo-EM could further confirm details about the orientation. The end result of the lab was purified proteins that were thoroughly purified and ready for EPR spin labels to be attached.