Production and characterization of chimeric Bst-like polymerases and their application in isothermal amplification combined with rapid RNA extraction methods using the example of the mumps virus

Introduction. Bst polymerase plays a key role in the rapid diagnosis of infectious diseases due to its unique biochemical properties and potential application in loop-mediated isothermal amplification (LAMP). Several analogs of Bst polymerase have been described in the literature; however, these enzymes have not been widely used in molecular diagnostics. The aim of the study is to obtain recombinant Bst and Btlv polymerases with the Sso7d domain and to test new possibilities for their application. Materials and methods. Expression constructs carrying the polymerase gene were obtained using standard genetic engineering methods. The target enzyme was produced in Escherichia coli cells. Purification was carried out using metal-affinity chromatography methods followed by dialysis and concentration. RNA-dependent DNA polymerase (reverse transcriptase) and DNA polymerase activities of the enzymes were determined using non-radioactive methods with fluorescent detection. The functional properties of the enzymes were assessed using the Amplisens SARS-CoV-2-IT reagent kit and a method designed for the detection of mumps virus RNA in biological material using the LAMP format combined with reverse transcription. Results. In the E. coli-based expression system, the following recombinant chimeric enzymes with displacing activity have been obtained: Bst_Sso7d, Bst_Sso7d_mut4 and Btlv_Sso7d. The developed cultivation and purification protocols allow for the production of enzymes in soluble form with a yield of up to 25% of the collected cell mass. Functional testing showed that in LAMP, the chimeric polymerases demonstrated similar activity to Bst polymerase without the Sso7d domain. At the same time, the Btlv_Sso7d polymerase exhibited increased reverse transcriptase activity and resistance to inhibitors. Conclusion. The obtained chimeric polymerase Btlv_Sso7d, due to its improved properties, can be used in reagent kits for the diagnosis of infectious diseases by the LAMP method when using nucleic acid extraction methods.