Foamy virus mutant reverse transcriptase
The present invention relates to a highly processive reverse transcriptase having DNA polymerase activity and substantially reduced protease activity. More specifically, the invention relates to an isolated reverse transcriptase from foamy virus comprising a substantially inactivated protease. The invention also relates to vectors containing the gene and hosts transformed with the vector of the invention. Further, the invention relates to a method for producing reverse transcriptase having DNA polymerase activity and substantially reduced protease activity by expressing the reverse transcriptase genes of the present invention in a recombinant host. Methods are also provided for producing cDNA from polynucleotides using the highly processive reverse transcriptase of the invention. Kits for the preparation of cDNA from RNA comprising the highly processive reverse transcriptase of the invention are also provided.
1. An isolated nucleic acid encoding a polyprotein consisting of foamy virus protease-reverse transcriptase having substantially reduced protease activity as compared with wild-type protease-reverse transcriptase and a highly processive reverse transcriptase, wherein the foamy virus protease-reverse transcriptase polyprotein comprises a mutation in the aspartic acid residue in the protease catalytic site, and wherein the highly processive reverse transcriptase can produce a nucleotide product of more than 600 basepairs in 10 minutes.
2. The nucleic acid of claim 1 , wherein the nucleic acid encodes a modified simian, cow, or human foamy virus protease-reverse transcriptase.
3. A vector comprising a nucleic acid encoding a polyprotein consisting of foamy virus protease-reverse transcriptase having substantially reduced protease activity as compared with wild-type protease-reverse transcriptase and a highly processive reverse transcriptase, wherein the foamy virus protease-reverse transcriptase polyprotein comprises a mutation in the aspartic acid residue in the protease catalytic site, and wherein the highly processive reverse transcriptase can produce a nucleotide products of more than 600 basepairs in 10 minutes.
4. A recombinant host cell comprising a vector of claim 3 .
5. A kit for the preparation of cDNA comprising an isolated foamy virus protease-reverse transcriptase polyprotein having a highly processive reverse transcriptase activity, and substantially reduced protease activity as compared with wild-type protease-reverse transcriptase, wherein the foamy virus protease-reverse transcriptase polyprotein comprises a mutation in the aspartic acid residue in the protease active site, a primer, an oligonucleotide template, deoxyribonucleotide bases, buffers sufficient for reverse transcription and a container and wherein the highly processive reverse transcriptase can produce a nucleotide product of more than 600 basepairs in 10 minutes.