2015年9月
Preparation and characterization of the RNase H domain of Moloney murine leukemia virus reverse transcriptase
PROTEIN EXPRESSION AND PURIFICATION
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- 巻
- 113
- 号
- 開始ページ
- 44
- 終了ページ
- 50
- 記述言語
- 英語
- 掲載種別
- 研究論文(学術雑誌)
- DOI
- 10.1016/j.pep.2015.04.012
- 出版者・発行元
- ACADEMIC PRESS INC ELSEVIER SCIENCE
Moloney murine leukemia virus reverse transcriptase (MMLV RT) contains fingers, palm, thumb, and connection subdomains as well as an RNase H domain. The DNA polymerase active site resides in the palm subdomain, and the RNase H active site is located in the RNase H domain. The RNase H domain contains a positively charged alpha-helix called the C helix (H(594)GEIYRRR(601)), that is thought to be involved in substrate recognition. In this study, we expressed three versions of the RNase H domain in Escherichia coil, the wild-type domain (WT) (residues Ile498-Leu671) and two variants that lack the regions containing the C helix (Ile593-Leu603 and Gly595-Thr605, which we called Delta C1 and Delta C2, respectively) with a strep-tag at the N-terminus and a deca-histidine tag at the C-terminus. These peptides were purified from the cells by anion-exchange, Ni2+ affinity, and Strep-Tactin affinity column chromatography, and then the tags were removed by proteolysis. In an RNase H assay using a 25-bp RNA-DNA heteroduplex, WT, Delta C1, and Delta C2 produced RNA fragments ranging from 7 to 16 nucleotides (nt) whereas the full-length MMLV RT (Thr24-Leu671) produced 14-20-nt RNA fragments, suggesting that elimination of the fingers, palm, thumb, and connection subdomains affects the binding of the RNase H domain to the RNA-DNA heteroduplex. The activity levels of WT, Delta C1, and Delta C2 were estimated to be 1%, 0.01%, and 0.01% of full-length MMLV RT activity, indicating that the C helix is important, but not critical, for the activity of the isolated RNase H domain. (C) 2015 Elsevier Inc. All rights reserved.
- リンク情報
- ID情報
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- DOI : 10.1016/j.pep.2015.04.012
- ISSN : 1046-5928
- eISSN : 1096-0279
- Web of Science ID : WOS:000356113300007