Marathon RT purification from recombinant E. coli via affinity chromatography
Board Location: #130
Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Session: 2
Stephanie Cruz - University of California, Santa Cruz
Co-Author(s): Elizabeth Wang, University of California, Santa Cruz, Santa Cruz; Melissa Jurica
Marathon reverse transcriptase (RT) possesses superior intrinsic processivity under conditions of excess RNA template. Additionally, the error frequency of MarathonRT is comparable to other high-performance reverse transcriptases. We sought to find a method for purifying MarathonRT from recombinant E. coli to utilize it as a future research tool. First, we raised a recombinant E. coli culture containing a plasmid bioengineered to induce the expression of MarathonRT. MarathonRT was also engineered to contain His and SUMO affinity tags. Before induction with IPTG to initiate transcription, the culture was incubated until an OD600 reading of 0.5-1 was reached. After sonification to break apart the cell membranes, and centrifugation to isolate soluble proteins, the protein was purified with His-Spin immobilized metal ion affinity chromatography (IMAC). It was then eluted with imidazole-containing buffers. We analyzed the product yield via SDS-Page gel electrophoresis and UV spectrophotometry. A significant amount of protein was detected in the elution samples, but the main band of protein was around 55 kDa, lower than the expected 62.4 kDa that MarathonRT should travel. In the future, it is necessary to analyze the identity of the purified protein and find the cause of the smaller size of the purified protein. We can also optimize the purification method to utilize it as a research tool.
Funder Acknowledgement(s): Funded by :the National Institutes of Health (NIH),the National Science Foundation (NSF), the University Office of the President (UCOP), and private donors.
Faculty Advisor: Melissa Jurica, mjurica@ucsc.edu
Role: I participated in the cultivation of a bioengineered plasmid that was ordered online wherein I subsequently induced the expression of Marathon RT using Isopropyl β-D-1-thiogalactopyranoside (IPTG).My responsibilities encompassed the comprehensive purification of the targeted protein through the implementation of affinity chromatography. Moreover, I conducted sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and ultraviolet (UV) spectrometry analyses on the obtained samples.