Instant Taq: Rapid Autoinducible Expression and Chromatography-free Purification of Taq polymerase

Abstract

AbstractDNA modifying enzymes are ubiquitous reagents in synthetic biology. Producing these enzymes often requires large culture volumes, purified nucleases and chromatographic separations to make enzymes of necessary quality. We sought to leverage synthetic biology tools to develop engineered strains allowing for not only the production but rapid purification of these reagents. Toward this goal, we report an E. coli strain enabling the rapid production and purification of Taq polymerase. The method relies on 1) autoinducible expression achieving high protein titers, 2) autolysis and auto DNA/RNA hydrolysis via lysozyme and a mutant benzonase™, and 3) heat denaturation under reducing conditions to precipitate contaminating proteins including the mutant benzonase™. Taq polymerase is obtained at high purities (>95% pure by SDS-PAGE) and is readily usable in standard reactions. The method takes less than 1 hour of hands-on time, does not require special equipment, expensive reagents or affinity purification. We expect this simple methodology and approach will improve access not only to Taq polymerase but to numerous additional commonly utilized reagent proteins.HighlightsProtein titers ~ 1g/L achieved in 20 mL shakeflasks.4 mg of purified Taq (corresponding to 5,000 Units, or 4,000 PCR reactions) per 20 mL shake flask.Instant Taq is equivalent to commercial preparations in routine PCR