TY - JOUR
T1 - Expression of the thermostable Moloney murine leukemia virus reverse transcriptase by silkworm-baculovirus expression system
AU - Yano, Takumi
AU - Lee, Jae Man
AU - Xu, Jian
AU - Morifuji, Yoshiki
AU - Masuda, Akitsu
AU - Hino, Masato
AU - Morokuma, Daisuke
AU - Fujita, Ryosuke
AU - Takahashi, Masateru
AU - Kusakabe, Takahiro
AU - Mon, Hiroaki
N1 - KAUST Repository Item: Exported on 2020-10-01
Acknowledgements: We 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).
PY - 2019/2/26
Y1 - 2019/2/26
N2 - 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.
AB - 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.
UR - http://hdl.handle.net/10754/652964
UR - https://www.sciencedirect.com/science/article/pii/S1226861519300159
UR - http://www.scopus.com/inward/record.url?scp=85062422843&partnerID=8YFLogxK
U2 - 10.1016/j.aspen.2019.02.008
DO - 10.1016/j.aspen.2019.02.008
M3 - Article
SN - 1226-8615
VL - 22
SP - 453
EP - 457
JO - Journal of Asia-Pacific Entomology
JF - Journal of Asia-Pacific Entomology
IS - 2
ER -