このアイテムのアクセス数: 440

このアイテムのファイル:
ファイル 記述 サイズフォーマット 
s10529-012-0904-9.pdf362.1 kBAdobe PDF見る/開く
タイトル: Improving the thermal stability of avian myeloblastosis virus reverse transcriptase α-subunit by site-directed mutagenesis.
著者: Konishi, Atsushi
Yasukawa, Kiyoshi  kyouindb  KAKEN_id
Inouye, Kuniyo
著者名の別形: 保川, 清
キーワード: cDNA synthesis
Avian myeloblastosis virus
Reverse transcriptase
Site-directed mutagenesis
Stability
発行日: Jul-2012
出版者: Springer Science+Business Media B.V.
誌名: Biotechnology letters
巻: 34
号: 7
開始ページ: 1209
終了ページ: 1215
抄録: Avian myeloblastosis virus reverse transcriptase (AMV RT) is a heterodimer consisting of a 63 kDa α-subunit and a 95 kDa β subunit. Moloney murine leukaemia virus reverse transcriptase (MMLV RT) is a 75 kDa monomer. These two RTs are the most extensively used for conversion of RNA to DNA. We previously developed several mutations that increase the thermostability of MMLV RT and generated a highly stable MMLV RT variant E286R/E302K/L435R/D524A by combining three of them (Glu286→Arg, Glu302→Lys, and Leu435→Arg) and the mutation to abolish RNase H activity (Asp524→Ala) [Yasukawa et al. (2010) J Biotechnol 150:299-306]. To generate a highly stable AMV RT variant, we have introduced the triple mutation of Val238→Arg, Leu388→Arg, and Asp450→Ala into AMV RT α-subunit and the resulted variant V238R/L388R/D450A, was expressed in insect cells and purified. The temperature decreasing the initial activity by 50 %, measured over 10 min, of the variant with or without template primer (T/P), poly(rA)-p(dT)(15), was 50 °C; for the wild-type AMV RT α-subunit (WT) this was 44 °C. The highest temperature at which the variant exhibited cDNA synthesis activity was 64 °C; the WT was 60 °C. A highly stable AMV RT α-subunit is therefore generated by the same mutation strategy as applied to MMLV RT and that positive charges are introduced into RT at positions that have been implicated to interact with T/P by site-directed mutagenesis.
著作権等: The final publication is available at www.springerlink.com
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/2433/157247
DOI(出版社版): 10.1007/s10529-012-0904-9
PubMed ID: 22426840
出現コレクション:学術雑誌掲載論文等

アイテムの詳細レコードを表示する

Export to RefWorks


出力フォーマット 


このリポジトリに保管されているアイテムはすべて著作権により保護されています。