抄録
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.
| 元の言語 | 英語 |
|---|---|
| ページ（範囲） | 453-457 |
| ページ数 | 5 |
| ジャーナル | Journal of Asia-Pacific Entomology |
| 巻 | 22 |
| 発行部数 | 2 |
| DOI | |
| 出版物ステータス | 出版済み - 6 1 2019 |
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All Science Journal Classification (ASJC) codes
- Insect Science
これを引用
Expression of the thermostable Moloney murine leukemia virus reverse transcriptase by silkworm-baculovirus expression system. / Yano, Takumi; Lee, Man; Xu, Jian; Morifuji, Yoshiki; Masuda, Akitsu; Hino, Masato; Morokuma, Daisuke; Fujita, Ryosuke; Takahashi, Masateru; Kusakabe, Takahiro; Mon, Hiroaki.
：: Journal of Asia-Pacific Entomology, 巻 22, 番号 2, 01.06.2019, p. 453-457.研究成果: ジャーナルへの寄稿 › 記事
}
TY - JOUR
T1 - Expression of the thermostable Moloney murine leukemia virus reverse transcriptase by silkworm-baculovirus expression system
AU - Yano, Takumi
AU - Lee, 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
PY - 2019/6/1
Y1 - 2019/6/1
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://www.scopus.com/inward/record.url?scp=85062422843&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85062422843&partnerID=8YFLogxK
U2 - 10.1016/j.aspen.2019.02.008
DO - 10.1016/j.aspen.2019.02.008
M3 - Article
VL - 22
SP - 453
EP - 457
JO - Journal of Asia-Pacific Entomology
JF - Journal of Asia-Pacific Entomology
SN - 1226-8615
IS - 2
ER -