Increase in thermal stability of Moloney murine leukaemia virus reverse transcriptase by site-directed mutagenesis

Abstract

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.

Stabilization of Moloney murine leukemia virus reverse transcriptase by site-directed mutagenesis of surface residue Val433

“…3 also indicates, the stabilities of MM7 and MM8 were similar to those of MM3 and MM4, which we generated previously. 8) The specific activities of the enzyme preparations were 31,000 units/mg for MM3, 35,000 units/mg for MM4, 33,000 units/mg for MM7, and 25,000 units/mg for MM8, almost the same.…”

“…7) We increased thermostability by introducing positive charges by site-directed mutagenesis at positions that have been implicated in interaction with the templateprimer (T/P), and generated highly stable multiple variants, E286R/E302K/L435R (MM3) and E286R/E302K/ L435R/D524A (MM4). 8) Their T 50 values, of MM3 and MM4, were 54 and 56°C, respectively, higher than that of WT (44°C). 8) We increased the thermostability of avian myeloblastosis virus (AMV) RT by the same strategy.…”

“…8) Their T 50 values, of MM3 and MM4, were 54 and 56°C, respectively, higher than that of WT (44°C). 8) We increased the thermostability of avian myeloblastosis virus (AMV) RT by the same strategy. 9,10) It is generally thought that surface hydrophobic residues decrease protein stability, but according to a summary of characterization of about 700 variants of the phage T4 lysozyme, 11) most surface residues contribute little to stability and the number of stabilizing mutations of surface residues is small, suggesting that the chances that one increases the thermostability of MMLV RT by replacing a surface hydrophobic residue with a charged one are low.…”

“…We think that the thermostable multiple MMLV RT variants that we 8) and others 6,7) have generated and which we generated in this study are more valuable for various research projects, including transcriptome analysis and clinical diagnosis, than wild-type MMLV RT. However, without a precise comparison of fidelity and processivity, it is difficult to determine which variant is the most suitable.…”

“…However, without a precise comparison of fidelity and processivity, it is difficult to determine which variant is the most suitable. To address this, further characterization of the thermostable variants generated previously (MM3 and MM4) 8) and in this study (MM7 and MM8) by misincoporation, mispair extension, and primer extension assay 17,18) is currently in progress.…”