To characterize Moloney murine leukemia virus (MMLV) reverse transcriptases (RTs) expressed in a cell-free system and in Escherichia coli.
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 A degrees C for 10 min, retained DNA polymerase activity, while WT, held at 54 A degrees C for 10 min, lost this activity. In the cDNA synthesis reaction (0.5 mu l) 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.
MMLV RT expressed in the cell-free system is indistinguishable from that expressed in E. coli.