Fina Amreta Laksmi, Eva Agustriana, Isa Nuryana, Rike Rachmayati, Kartika Sari Dewi, Asta Ismadara
DNA polymerase from Geobacillus stearothermophilus (previously Bacillus stearothermophilus), Bst polymerase, is an enzyme that used in loop-mediated isothermal amplification (LAMP) assay, that has proven to be a useful assay for the detection of COVID-19. Nevertheless, despite its usefulness, development of Bst polymerase in Indonesia is still scarce. For this reason, codon-optimized gene encoding Bst polymerase with the substitutions of one residue was employed. In addition, the experimental condition for expression of Bst polymerase was optimized to obtain higher expression of Bst polymerase and was subsequently purified by affinity chromatography. The enzyme was produced in Escherichia coli BL21 star (DE3) using pD871 expression vector, employing rhamnose-based promoter system. Optimum condition for expression was investigated by varying the induction temperature, L-rhamnose concentration, post induction incubation time, and initial pre-induction optical density (OD600). Our investigation showed that Bst polymerase exhibited higher expression level when it was induced at 37 °C with an initial pre-induction OD600 of 0.6-0.8. Moreover, induction for 12 h and addition of 2 mM L-rhamnose was found to be the optimum condition for expression. The improvement of protein yield as much as 2.9-old was achieved by applying optimized conditions. Bst polymerase was also successfully purified by eliminating other non-targeted bands. © 2023 Author(s).
Research Center for Biotechnology, Research Organization of Life Sciences, National Research and Innovation Agency (BRIN), West Java, Cibinong, 16911, Indonesia; Chemistry Department, Faculty of Mathematics and Science, State University of Malang, Malang, Indonesia