title: Expression of moloney murine leukemia virus reverse transcriptase in a cell-free protein expression system.
abstract: OBJECTIVE
  To characterize Moloney murine leukemia virus (MMLV) reverse transcriptases (RTs) expressed in a cell-free system and in Escherichia coli.
  
  
  RESULTS
  We previously expressed MMLV RT using an E. coli expression system and generated a highly thermostable quadruple variant MM4 (E286R/E302K/L435R/D524A) by site-directed mutagenesis. In this study, we expressed the wild-type MMLV RT (WT) and MM4 using a cell-free protein expression system from insect cells. WT exhibited DNA polymerase and RNase H activities, while MM4, in which the catalytic residue for RNase H activity, Asp524 is changed into Ala, exhibited only DNA polymerase activity. MM4, when held at 60 °C for 10 min, retained DNA polymerase activity, while WT, held at 54 °C for 10 min, lost this activity. In the cDNA synthesis reaction (0.5 μl) in which WT or MM4 were exposed to various temperatures and amounts of target RNA in a microarray chip, MM4 exhibited higher thermostability than WT.
  
  
  CONCLUSIONS
  MMLV RT expressed in the cell-free system is indistinguishable from that expressed in E. coli.
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
authors.forename: Yuta
authors.initials: Y
authors.lastname: Katano
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Hisayoshi
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
authors.forename: Ikumi
authors.initials: I
authors.lastname: Kuze
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Okano
authors.affiliation: Okinawa National College of Technology, Nago, Japan.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Ito
authors.affiliation: Saitama-Bio-Monodukuri-Network Research Organization, Saitama University, Saitama, Japan.|National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Nishigaki
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
authors.forename: Teisuke
authors.initials: T
authors.lastname: Takita
authors.affiliation: Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan. yasukawa@kais.kyoto-u.ac.jp.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Yasukawa
mesh_terms: D000818:Animals; D002474:Cell-Free System; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D008979:Moloney murine leukemia virus / Q000201:enzymology*; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
chemical_list: D012194:RNA-Directed DNA Polymerase
keywords: Cell-free expression; MMV; Moloney murine leukemia virus; Reverse transcriptase; Thermostability