Preliminary expression, purification, and functional characterization of the protein 2O14, a putative hydrolase.
Start Date
April 2024
Location
2nd floor - Library
Abstract
BASIL (The Biochemistry Authentic Scientific Inquiry Laboratory Community) is a course-based research experience (CURE) that uses computational techniques and traditional laboratory methods to assign functions to proteins. The goal of this research included expressing, purifying, and characterizing the function of the protein 2O14, which was identified in the bacterium Bacillus subtilis. Prior to our study, the X-ray crystal structure of 2O14 was published. However, the function of 2O14 remains largely unknown. We used the in silico bioinformatic programs SPRITE, DALI, InterPro, and BLAST to compare the structure of 2O14 to other proteins of known function. The results of these in silico models suggest that 2O14 is a hydrolase that may cleave ester and amide bonds. Next, we overexpressed 2O14 in Escherichia coli using the autoinduction method. Then we used nickel affinity resin to isolate 2O14 from all other bacterial proteins. SDS-PAGE revealed about 100 mL of protein at approximately 90% purity. A280 absorbance was used to determine a final concentration of 4.65 µM. Preliminary kinetic assays using para-nitrophenyl acetate as a substrate revealed hydrolase activity. We then optimized the kinetic assays testing various pH’s and substrates. Future in silico experiments will include ligand docking to explore other possible substrates. We also plan to perform thermal shift assays to determine whether thermal stability correlates to protein activity.
Preliminary expression, purification, and functional characterization of the protein 2O14, a putative hydrolase.
2nd floor - Library
BASIL (The Biochemistry Authentic Scientific Inquiry Laboratory Community) is a course-based research experience (CURE) that uses computational techniques and traditional laboratory methods to assign functions to proteins. The goal of this research included expressing, purifying, and characterizing the function of the protein 2O14, which was identified in the bacterium Bacillus subtilis. Prior to our study, the X-ray crystal structure of 2O14 was published. However, the function of 2O14 remains largely unknown. We used the in silico bioinformatic programs SPRITE, DALI, InterPro, and BLAST to compare the structure of 2O14 to other proteins of known function. The results of these in silico models suggest that 2O14 is a hydrolase that may cleave ester and amide bonds. Next, we overexpressed 2O14 in Escherichia coli using the autoinduction method. Then we used nickel affinity resin to isolate 2O14 from all other bacterial proteins. SDS-PAGE revealed about 100 mL of protein at approximately 90% purity. A280 absorbance was used to determine a final concentration of 4.65 µM. Preliminary kinetic assays using para-nitrophenyl acetate as a substrate revealed hydrolase activity. We then optimized the kinetic assays testing various pH’s and substrates. Future in silico experiments will include ligand docking to explore other possible substrates. We also plan to perform thermal shift assays to determine whether thermal stability correlates to protein activity.
