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タイトル: Increase in thermal stability of Moloney murine leukaemia virus reverse transcriptase by site-directed mutagenesis.
著者: Yasukawa, Kiyoshi  kyouindb  KAKEN_id
Mizuno, Masaki
Konishi, Atsushi
Inouye, Kuniyo
著者名の別形: 保川, 清
キーワード: cDNA synthesis
Moloney murine leukaemia virus
Reverse transcriptase
Site-directed mutagenesis
Stability
発行日: Nov-2010
出版者: Elsevier B.V.
誌名: Journal of biotechnology
巻: 150
号: 3
開始ページ: 299
終了ページ: 306
抄録: We hypothesized that the thermal stability of Moloney murine leukaemia virus reverse transcriptase (MMLV RT) will increase with increases in its ability to bind with a template-primer (T/P). To test this hypothesis, we introduced positive charges into MMLV RT by site-directed mutagenesis at positions that have been implicated in the interaction with T/P. Thirty-six variants were constructed in which one of the twelve residues (Glu69, Gln84, Asp108, Asp114, Glu117, Glu123, Asp124, Glu286, Glu302, Trp313, Leu435, and Asn454) was replaced with Lys, Arg, or Ala, and these were expressed in Escherichia coli. In about half of these 36 variants, thermal inactivation at 50°C was reduced in the presence of the T/P, which suggested that this strategy was effective at stabilizing MMLV RT. We next combined three of the 36 mutations, Glu286→Ala, Glu302→Lys, and Leu435→Arg, and the mutation, Asp524→Ala, which is known to abolish the RNase H activity and increase the stability. Temperatures of 54 and 56°C reduced the initial reverse transcription activity by 50% over a 10-min incubation in the triple variant E286R/E302K/L435R and quadruple variant E286R/E302K/L435R/D524A, respectively. These temperatures were higher than that observed for WT (45°C). The highest temperature at which the triple and quadruple variants exhibited cDNA synthesis activity was 60°C, which was again higher than for WT (54°C). Thus, highly stable MMLV RT variants were generated by this mutation strategy.
著作権等: © 2010 Elsevier B.V.
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/2433/131848
DOI(出版社版): 10.1016/j.jbiotec.2010.09.961
PubMed ID: 20933548
出現コレクション:学術雑誌掲載論文等

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