Mutant reverse transcriptase with increased thermal stability as well as products, methods and uses involving the same
The present invention relates to a mutant reverse transcriptase (RT) with increased thermal stability relative to the wildtype, a nucleic acid encoding the mutant RT, a cell comprising the mutant RT or the nucleic acid, a kit comprising the mutant RT, the use of the mutant RT for cDNA synthesis, method for reverse transcription of RNA comprising synthesizing cDNA with the use of the mutant RT and a method for detecting an RNA marker in a sample with the use of the mutant RT.
1. A method for reverse transcription of a mRNA, the method comprising synthesizing a cDNA from the mRNA using a mutant reverse transcriptase (RT), the mutant RT consisting of
i) an amino acid sequence that has only six amino acid substitutions of SEQ ID NO: 1, wherein, in SEQ ID NO:1,
Ala at position 32 is substituted with Val (A32V);
Leu at position 72 is substituted with Arg (L72R);
Glu at position 286 is substituted with Arg (E286R);
Glu at position 302 is substituted with Lys (E302K);
Trp at position 388 is substituted with Arg (W388R); and
Leu at position 435 is substituted with Arg (L435R),
wherein the mutant RT exhibits a reverse transcriptase activity, and wherein the mutant RT also has an increased thermal stability relative to a mutant MM3, wherein the mutant MM3 has an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 1 and only has three amino acid substitutions of SEQ ID NO: 1, wherein, in SEQ ID NO:1, Glu at position 286 is substituted with Arg (E286R), Glu at position 302 is substituted with Lys (E302K) and Leu at position 435 is substituted with Arg (L435R).
2. The method of claim 1 , wherein the reverse transcriptase activity of the mutant RT is at least 50% of the reverse transcriptase activity of the wildtype RT.
3. The method of claim 1 , wherein the mutant RT is capable of fusing to another protein.
4. The method of claim 1 , wherein the mutant RT consists of SEQ ID NO:2.