COVID-19 pandemic has established the importance of molecular biology-based detection systems for confirmatory diagnosis of diseases. Loop-mediated isothermal amplification (LAMP) is a molecular technique used for early detection of various infections caused by bacterial and viral agents. Bst DNA polymerase is a critical reagent used in LAMP. In order to produce this reagent in bulk, a batch fermentation process was developed in a bioreactor. E. coli is a bacterial host expression system commonly used for cost-effective production of recombinant proteins. Here, recombinant Bst DNA polymerase was cloned, and expressed in E. coli. Cultures were grown till mid-log phase and induced with isopropyl-beta-D-thiogalactopyranoside for recombinant protein expression. Effect of inducer concentration and cultivation media was studied in order to enhance the expression of recombinant Bst DNA polymerase. After process optimization, batch fermentation process was done for scale-up of the enzyme production. After cell harvesting and cell disruption, immobilized metal affinity chromatography was carried out to purify recombinant Bst DNA polymerase and resulted in 13.2 mg protein per liter of bioreactor culture. Purity of this enzyme was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. LAMP further confirmed its utility for confirmatory diagnosis of diseases. The present study established that the indigenously produced Bst DNA polymerase may be used for the diagnosis of diseases using LAMP in laboratories during any disease outbreak scenario, as well as in food safety testing and environmental monitoring in agriculture and forestry.
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