Stabilized reverse transcriptase fusion proteins
Stabilized reverse transcriptase fusion proteins including a thermostable reverse transcriptase connected to a stabilizer protein are described. Attaching the stabilizer protein to the thermostable reverse transcriptase stabilizes the fusion protein and can aid in its purification, provide increased solubility, allow for longer storage, or allow the fusion protein to be used under more rigorous conditions such as higher temperature. The stabilized reverse transcriptase fusion protein can also include a linker between the stabilizer protein and the thermostable reverse temperature. The stabilized reverse transcriptase fusion proteins are suitable for use in nucleic acid amplification methods such as the reverse transcription polymerase chain reaction and other applications involving cDNA synthesis.
1. A method of reverse transcription, comprising carrying out reverse transcription of an RNA template using a stabilized reverse transcriptase fusion protein comprising a group-II intron-derived reverse transcriptase connected at its N-terminus by a linker peptide to the C-terminus of a stabilizer protein comprising 50 or more amino acids, wherein the fusion protein exhibits increased solubility and stability in solution.
2. The method of claim 1 , wherein the reverse transcriptase comprises a polypeptide having at least 85% amino acid sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.
3. The method of claim 1 , wherein the stabilized reverse transcriptase fusion protein comprises an amino acid sequence with at least 85% amino acid sequence identity to a sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.
4. The method of claim 1 , wherein the solubility-enhancing stabilizer protein comprises a maltose binding protein or an N-utilization substance A protein.
5. The method of claim 1 , wherein the solubility-enhancing stabilizer protein does not fold into long-lived misfolded intermediates.
6. The method of claim 1 , wherein the linker peptide is a non-cleavable linker peptide.
7. The method of claim 1 , wherein the linker peptide is a rigid linker peptide.
8. The method of claim 1 , wherein the reverse transcription is carried out with an error frequency of 2.0×10 −5 or less at a temperature from about 45° to about 65° C.
9. The method of claim 1 , wherein the solubility-enhancing stabilizer protein includes an independent folding domain.
10. The method of claim 7 , wherein the rigid linker peptide consists of 3 to 5 amino acids.
11. The method of claim 10 , wherein the rigid linker peptide consists of SEQ ID NO: 12 or SEQ ID NO: 18.
12. A method of reverse transcription, comprising carrying out reverse transcription of an RNA template using a stabilized reverse transcriptase fusion protein comprising a group-II intron-derived reverse transcriptase connected at its N-terminus to the C-terminus of a stabilizer protein comprising 50 or more amino acids, wherein the fusion protein exhibits increased solubility and stability in solution.