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タイトル: Stabilization of human immunodeficiency virus type 1 reverse transcriptase by site-directed mutagenesis.
著者: Nishimura, Kosaku
Shinomura, Mayu
Konishi, Atsushi
Yasukawa, Kiyoshi  kyouindb  KAKEN_id
著者名の別形: 保川, 清
キーワード: HIV
Human immunodeficiency virus type 1
Reverse transcriptase
Site-directed mutagenesis
Thermostability
発行日: Dec-2013
出版者: Springer Netherlands
誌名: Biotechnology letters
巻: 35
号: 12
開始ページ: 2165
終了ページ: 2175
抄録: Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a heterodimer containing 66 kDa p66 and 51 kDa p51 subunits. We previously showed that HIV-1 group M (HIV-1 M) RT and HIV-1 group O (HIV-1 O) RT have higher affinities for dTTP and template-primer (T/P) than Moloney murine leukemia virus RT, which is currently used for cDNA synthesis, suggesting that they might also be useful for cDNA synthesis (Konishi et al. Appl Biochem Biotechnol 2013, 169:77-87). Here, we have increased the thermostability of both HIV-1 M RT and HIV-1 O RT by site-directed mutagenesis. The Asp443 → Ala mutation, which abolishes RNase H activity, was introduced into the p66 subunits of HIV-1 M RT and HIV-1 O RT. The temperatures that reduced the initial activity by 50 % of the resulting mutants, HIV-1 M p66D443A/p51 and HIV-1 O p66D443A/p51, were 44 and 52 °C, respectively, which were higher than those of wild-type HIV-1 M p66/p51 (42 °C) and HIV-1 O p66/p51 (48 °C). The highest temperature at which both HIV-1 M p66D443A/p51 and HIV-1 O p66D443A/p51 exhibited cDNA synthesis activity was 68 °C, which was higher than for the wild-type enzymes (62 and 66 °C, respectively).
著作権等: The final publication is available at Springer via http://dx.doi.org/10.1007/s10529-013-1321-4
This is not the published version. Please cite only the published version. この論文は出版社版でありません。引用の際には出版社版をご確認ご利用ください。
URI: http://hdl.handle.net/2433/192980
DOI(出版社版): 10.1007/s10529-013-1321-4
PubMed ID: 24078120
出現コレクション:学術雑誌掲載論文等

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