Experiences of Undergraduate Students Identifying Proteins of Unknown Function As Part of a Teaching Laboratory in a Biochemistry Course

Abstract

In the laboratory section of Biochemistry II at St. Mary's University, we are performing a series of experiments involving authentic scientific inquiry. The experiments' goal is to identify the functions of a handful of unknown proteins. The curriculum relies heavily on both in silico and in vitro biochemistry techniques. Faculty and students pick the proteins by looking through the PDB list of crystallized proteins that have no known function. Since these proteins have been crystallized, they have already been cloned and purified; we know we are likely to be able to isolate suitable quantities of the proteins for work. We ascertain ahead of time that their coding vectors are available to us from sources such as DNASU or AddGene. We analyze the sequences of potential proteins using internet programs such as Pfam, Dali, BLAST, and HHPred. Good alignment with characterized proteins allows us to make hypotheses for the function of the unknown ones. We use the PyMol plug‐in ProMol to look for arrangements of active site residues common to certain categories of enzymes. For example, we used the triad of serine, histidine, and aspartate from chymotrypsin to identify possible hydrolases. We use the three‐dimensional alignment function of the program Chimera to see whether our unknown proteins resemble the known proteins that are thought to be similar. We overexpress the unknown proteins in E. coli, and purify the enzymes taking advantage of their engineered tags. The spring 2016 class focused on and was successful in broadly identifying four unknown hydrolases. Of these, three were lipases and one was a nuclease. We are extending that success by purifying and assaying additional hydrolases in spring 2017. After every lab in the pilot semester students reported on their experiences, and they presented posters at a University research symposium. The student authors of this poster will either have performed the pilot semester (SM) or be involved in the second round of the curriculum (JB, JB, and EV). They will report on their experiences, their experiments, their findings and whether this innovative teaching lab affected their attitudes toward the inquiry process.Support or Funding InformationThis work is funded by NSF DUE‐1502720.