T4 DNA LIGASE: DETERMINING THE OPTIMAL SYSTEM FOR INDUSTRIAL EXPRESSION AND PURIFICATION

Abstract

Molecular biology enzymes are necessary tools for the generation of recombinant DNA molecules. Making these enzymes available at the lowest possible cost will enable molecular biology laboratories to increase production. In order to achieve this, molecular biology enzyme supply companies must determine ways to produce these enzymes at the lowest possible cost. The production of T4 DNA ligase was evaluated using E. coli, B. megaterium, and the baculovirus expression systems. Expression and sonication studies were performed to optimize enzyme yield. Final optimal conditions were used to generate 1L cell pellets for purification. Protein assays confirm a higher yield of T4 DNA ligase from the baculovirus system than from E. coli or B. megaterium. It was hypothesized that the specific activity of purified T4 DNA ligase from each system would be similar. A novel technique using the Agilent 2100 Bioanalyzer was evaluated to determine ligase activity. The assay results indicate all three expression systems produced ligase of similar activity. The cost analysis, in conjunction with the yield indicates E. coli is the best system for recombinant T4 DNA ligase production at the 1L scale.