An enhanced activity and thermostability of chimeric Bst DNA polymerase for isothermal amplification applications.

Abstract

Loop-mediated isothermal amplification (LAMP) is a widely used method for clinical diagnosis, customs quarantine, and disease prevention. However, the low catalytic activity of Bst DNA polymerase has made it challenging to develop rapid and reliable point-of-care testing. Herein, we developed a series of Bst DNA polymerase mutants with enhanced activity by predicting and analyzing the activity sites. Among these mutants, single mutants K431D and K431E showed a 1.93- and 2.03-fold increase in catalytic efficiency, respectively. We also created a chimeric protein by fusing the DNA-binding domain of DNA ligase from Pyrococcus abyssi (DBD), namely DBD-K431E, which enabled real-time LAMP at high temperatures up to 73 ℃ and remained active after heating at 70 ℃ for 8h. The chimeric DBD-K431E remained active in the presence of 50 U/mL heparin, 10% ethanol, and up to 100mM NaCl, and showed higher activity in 110mM (NH4)2SO4, 110mM KCl, and 12mM MgSO4. Notably, it generated a fluorescence signal during the detection of Salmonella typhimurium at 2 × 102 ag/μL of genomic DNA and 1.24CFU/mL of bacterial colony, outperforming the wild type and the commercial counterpart Bst 2.0. Our results suggest that the DBD-K431E variant could be a promising tool for general molecular biology research and clinical diagnostics. KEY POINTS: • Residue K431 is probably a key site of Bst DNA polymerase activity • The chimeric DBD-K431E is more inhibitor tolerant and thermostable than Bst-LF • The DBD-K431E variant can detect Salmonella typhimurium at 102 ag/μL or 100CFU/mL.