Expression of the thermostable Moloney murine leukemia virus reverse transcriptase by silkworm-baculovirus expression system
Lee, Jae Man
KAUST DepartmentBiological and Environmental Sciences and Engineering (BESE) Division
Laboratory of DNA Replication and Recombination
Permanent link to this recordhttp://hdl.handle.net/10754/652964
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AbstractReverse transcriptase from Moloney murine leukemia virus (MMLVRT) is an RNA dependent DNA polymerase, which has been used as a fundamental tool for molecular biology and biotechnology. The secondary structures formed in the RNA templates decrease the accessibility of the reverse transcriptase to the RNA templates; it is important to unfold the RNA secondary structure by increasing the reaction temperature to perform the successful transcription. In this study, we applied silkworm baculovirus expression vector system (silkworm-BEVS) to mass-produce and purify the recombinant MMLVRTs with the N- or C- terminal tandem tags. We confirmed that both of the recombinant MMLVRT enzymes have intact DNA polymerase activity. It is notable that C- terminal tagged MMLVRT outperformed MMLVRT obtained from the E. coli expression system in terms of thermostability and sensitivity to low quantities of RNA template. Taken together, these results demonstrate that silkworm-BEVS is a promising alternative strategy to produce the functional and thermostable reverse transcriptase.
CitationYano T, Lee JM, Xu J, Morifuji Y, Masuda A, et al. (2019) Expression of the thermostable Moloney murine leukemia virus reverse transcriptase by silkworm-baculovirus expression system. Journal of Asia-Pacific Entomology 22: 453–457. Available: http://dx.doi.org/10.1016/j.aspen.2019.02.008.
SponsorsWe thank Dr. Imanishi (National Institute of Agrobiological Sciences, Japan) for kindly providing the NIAS-Bm-oyanagi2 (BmO2) cell line. This work was supported by Japan Science and Technology Agency (JST) for the Program for Creating Start-ups from Advanced Research and Technology (START Program).