(R4778) Expression of heterologous Polymerase n (PolN) using fusion partner protein maltose-binding protein

Ngoc H. Huynh (San Diego State University)

Polymerase n (PolN) is a poorly understood error-prone polymerase that is involved in DNA repair pathways. PolN-deficient cells are more sensitive to interstrand cross-links (ICL) agents, suggesting that PolN is involved in repairing ICL. PolN has also been linked to cancer, including amplification in neuroendocrine prostate cancers, but its role is unknown. One of the challenges that hinders PolN studies is the poor heterologous expression of PolN. Here we seek to systematically improve the expression and purification of PolN heterologously expressed in  E. coli. Plasmids with fusion partners that enhance high expression of insoluble proteins have been widely used. We employed the maltose-binding protein (MBP) as a solubility-enhanced protein and His tag to facilitate bacterial expression and purification. The Gateway plasmid was transformed to BL21 DE3 bacterial strain and the expression was induced by adding 0.5mM isopropyl β- d-1-thiogalactopyranoside (IPTG) after an OD600 of 0.6 was reached and kept constant at cold temperature overnight. The expression was optimized further to increase soluble protein production, including addition of arabinose and varying concentrations of both arabinose and IPTG. The induction temperature was kept at 37 degree Celsius and was kept constant for a duration of less than 10 hours. Finally, the cells were harvested and analyzed by SDS-PAGE to assess expression levels. The higher expression level was achieved at a cold temperature in the presence of both IPTG and 1% of arabinose. To achieve higher concentration of PolN harvested, we will increase the volume of cultures that will be expressed and purification will be done using affinity chromatography.

Support or Funding Information

This work was funded by a Research Scholar Grant, RSG-19-075-01-TBE, from the American Cancer Society (C.D.S.), National Institutes of Health R00 CA187594 (C.D.S.), U54CA132384 (SDSU) & U54CA132379 (UC San Diego), MARC 5T34GM008303 (SDSU), and IMSD 5R25GM058906 (SDSU), as well as the California Metabolic Research Foundation (SDSU).