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 transcriptase. 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 stabilized reverse transcriptase fusion protein comprising a thermostable group-II intron-derived reverse transcriptase connected at its N-terminus by a linker peptide to the C-terminus of a stabilizer protein including 50 or more amino acids, wherein the fusion protein exhibits increased solubility and stability in solution.
2. The stabilized reverse transcriptase fusion protein of claim 1 , wherein the thermostable reverse transcriptase is a Thermosynechococcus elongatus reverse transcriptase, or Geobacillus stearothermophilus reverse transcriptase.
3. The stabilized reverse transcriptase fusion protein of claim 1 , wherein the thermostable reverse transcriptase comprises a polypeptide with 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.
4. The stabilized reverse transcriptase fusion protein of claim 1 , wherein the stabilizer protein comprises an affinity protein or a solubility-enhancing protein.
5. The stabilized reverse transcriptase fusion protein of claim 4 , wherein the stabilizer protein comprises a maltose binding protein or an N-utilization substance A protein.
6. The stabilized reverse transcriptase fusion protein of claim 4 , wherein the stabilizer protein has been modified by replacing charged amino acids with uncharged amino acids.
7. The stabilized reverse transcriptase fusion protein of claim 1 , wherein the linker peptide is a non-cleavable linker peptide.
8. The stabilized reverse transcriptase fusion protein of claim 7 , wherein the non-cleavable linker peptide is a rigid linker peptide.
9. The stabilized reverse transcriptase fusion protein of claim 7 , wherein the linker peptide consists of 1 to 20 amino acids.
10. The stabilized reverse transcriptase fusion protein of claim 7 , wherein the linker peptide consists of 1 to 5 amino acids.
11. The stabilized reverse transcriptase fusion protein of claim 7 , wherein the linker peptide consists of 3 to 5 amino acids.
12. The stabilized reverse transcriptase fusion protein of claim 8 , wherein the rigid linker peptide consists of SEQ ID NO: 12.
13. The stabilized reverse transcriptase fusion protein of claim 1 , wherein the fusion protein has an amino acid sequence comprising a polypeptide 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.
14. The stabilized reverse transcriptase fusion protein of claim 1 , wherein the stabilized reverse transcriptase fusion protein is capable of carrying out reverse transcription with an error frequency of 2.0×10 −5 or less at a temperature from about 45° to about 65° C.
15. A stabilized reverse transcriptase fusion protein comprising a thermostable group-II intron-derived reverse transcriptase connected at its N-terminus by a linker peptide to the C-terminus of a stabilizer protein including 50 or more amino acids, wherein the fusion protein exhibits increased solubility and stability in solution.
16. The stabilized reverse transcriptase fusion protein of claim 1 , wherein the stabilizer protein includes an independent folding domain and/or does not fold into long-lived misfolded intermediates.