Expression of the thermostable Moloney murine leukemia virus reverse transcriptase by silkworm-baculovirus expression system

• Published in: Journal of Asia-Pacific entomology Vol. 22; no. 2; pp. 453 - 457
• Main Authors: Yano, Takumi, Lee, Jae Man, Xu, Jian, Morifuji, Yoshiki, Masuda, Akitsu, Hino, Masato, Morokuma, Daisuke, Fujita, Ryosuke, Takahashi, Masateru, Kusakabe, Takahiro, Mon, Hiroaki
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.06.2019; 한국응용곤충학회
• Subjects: Baculoviridae; Baculovirus expression system; biotechnology; DNA-directed DNA polymerase; enzyme activity; Escherichia coli; genetic vectors; MMLVRT; molecular biology; Murine leukemia virus; Reverse transcriptase; RNA; RNA-directed DNA polymerase; Silkworm; silkworms; temperature; thermal stability; transcription (genetics); 농수해양학; Baculovirus expression system; MMLVRT; Reverse transcriptase; Silkworm
• ISSN: 1226-8615; 1876-7990
• DOI: 10.1016/j.aspen.2019.02.008

Reverse 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. [Display omitted] •Moloney murine leukemia virus reverse transcriptase (MMLVRT) was expressed in silkworm-baculovirus expression vector system.•Recombinant MMLVRT enzymes showed intact RNA-dependent DNA polymerase activity.•C-terminal tagged MMLVRT showed higher thermostability than that of MMLVRT produced in E. coli.