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 group-II intron reverse transcriptase connected at its N-terminus 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 - 3 . (canceled)
4 . The stabilized reverse transcriptase fusion protein of claim 1 , wherein the group-II intron reverse transcriptase is a Lactococcus lactis reverse transcriptase, a Thermosynechococcus elongatus reverse transcriptase, or Geobacillus stearothermophilus reverse transcriptase.
5 . (canceled)
6 . The stabilized reverse transcriptase fusion protein of claim 1 , wherein the stabilizer protein comprises an affinity protein or a solubility-enhancing protein.
7 . The stabilized reverse transcriptase fusion protein of claim 6 , wherein the stabilizer protein comprises a maltose binding protein or an N-utilization substance A protein.
8 . The stabilized reverse transcriptase fusion protein of claim 6 , wherein the stabilizer protein has been modified by replacing charged amino acids with uncharged amino acids.
9 . The stabilized reverse transcriptase fusion protein of claim 1 , wherein the reverse transcriptase is connected to the stabilizer protein by a linker peptide.
10 . The stabilized reverse transcriptase fusion protein of claim 1 , wherein the linker peptide is a non-cleavable linker peptide.
11 . The stabilized reverse transcriptase fusion protein of claim 10 , wherein the non-cleavable linker peptide is a rigid linker peptide.
12 . The stabilized reverse transcriptase fusion protein of claim 10 , wherein the linker peptide consists of 1 to 20 amino acids.
13 . The stabilized reverse transcriptase fusion protein of claim 10 , wherein the linker peptide consists of 1 to 5 amino acids.
14 . The stabilized reverse transcriptase fusion protein of claim 10 , wherein the linker peptide consists of 3 to 5 amino acids.
15 . The stabilized reverse transcriptase fusion protein of claim 11 , wherein the rigid linker peptide consists of SEQ ID NO: 12.
16 . (canceled)
17 . The stabilized reverse transcriptase fusion protein of claim 1 , wherein the stabilized reverse transcriptase fusion protein is capable of carrying out reverse transcription of an RNA template with processivity or fidelity greater than that of SuperScript III.
18 . A method for preparing a cDNA from an RNA molecule, comprising the steps of:
(a) adding a primer nucleotide sequence to the RNA molecule
(b) incubating the RNA molecule in the presence of one or more deoxy or dideoxyribonucleoside triphosphates and a stabilized reverse transcriptase fusion protein comprising a group-II intron reverse transcriptase connected at its N-terminus 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 under conditions sufficient to synthesize a cDNA molecule complementary to all or a portion of the RNA molecule.
19 . The method of claim 18 , wherein the group II intron reverse transcriptase is connected to the stabilizer protein by a linker peptide.
20 . The method of claim 18 , wherein reverse transcription is performed within a temperature range where the RNA has a substantially decreased amount of obstructing stable secondary or tertiary structure.
21 . The method of claim 19 , wherein the linker peptide is a non-cleavable linker peptide.
22 . The method of claim 19 , wherein the group II intron 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, the linker consists of 1 to 20 amino acids, and the stabilizer protein comprises an affinity protein or a solubility-enhancing protein.
23 . The method of claim 18 , wherein the reverse transcription is performed with an error frequency of 2.0×10 −5 or less at a temperature from about 45° C. to about 65° C.
24 . A DNA expression vector for producing a stabilized reverse transcriptase fusion protein, comprising an isolated nucleic acid that encodes a polypeptide comprising a group II intron reverse transcriptase 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.
25 . A method of producing a stabilized reverse transcriptase fusion protein, comprising the steps of
(a) culturing a host cell comprising the DNA expression vector of claim 24 ;
(b) expressing the stabilized reverse transcriptase fusion protein encoded by the DNA expression vector; and
(c) isolating the stabilized reverse transcriptase fusion protein from the host cell.
26 . 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.