Mutant T7 polymerases

Patent metadata

Field	Value
Publication number	US-9062292-B2
Application number	US-80775110-A
Country	US
Kind code	B2
Filing date	Sep 13, 2010
Priority date	Sep 13, 2010
Publication date	Jun 23, 2015
Grant date	Jun 23, 2015

Abstract

Provided are mutant polymerases that comprise a deletion of at least four amino acids among the amino acids at positions corresponding to 167-174 of SEQ ID NO:1. Also provided are mutant polymerases having greater resistance to 30 mM NaCl, 7.5 mM phosphate, or 20 μg/ml single stranded DNA than a wild-type T7 RNA polymerase having SEQ ID NO:1 or a wild-type T3 RNA polymerase having SEQ ID NO:3. Nucleic acids comprising a nucleotide sequence encoding any of the above mutant polymerases are also provided, as are vectors comprising those nucleic acids and host cells transformed with the vectors Additionally, methods of amplifying mRNA using the mutant polymerases described herein are also provided. Further, compositions comprising any of the mutant polymerases described herein, and a reagent at a concentration that is inhibitory to wild-type T7 RNA polymerase is provided.

First claim

What is claimed is:

1. A synthetically generated polymerase selected from (i) a protein having an amino acid sequence that is at least 90% homologous to the amino acid sequence of the polymerase of SEQ ID NO: 1 and a deletion of the 8 amino acids corresponding to residues 167-174 of SEQ ID NO: 1 and (ii) a protein having an amino acid sequence that is at least 90% homologous to the amino acid sequence of the polymerase of SEQ ID NO:3 and a deletion of the 8 amino acids corresponding to residues 168-175 of SEQ ID NO: 3.

2. The polymerase of claim 1 , having an amino acid sequence that is at least 95% homologous to the polymerase of SEQ ID NO: 1 or the polymerase of SEQ ID NO:3.

3. The polymerase of claim 1 , having an amino acid sequence that is at least 98% homologous to SEQ ID NO:1 or SEQ ID NO:3.

4. The polymerase of claim 1 , having an amino acid sequence that is at least 99% homologous to SEQ ID NO:1 or SEQ ID NO:3.

5. A polymerase having the amino acid sequence of SEQ ID NO: 2.

6. The polymerase of claim 1 , having greater resistance to 30 mM NaCl, 7.5 mM phosphate, 7.5 mM pyrophosphate, or 20 μg/ml single stranded DNA than the wild-type T7 RNA polymerase having the sequence of SEQ ID NO:1 or the wild-type T3 RNA polymerase having the sequence of SEQ ID NO:3.

7. The polymerase of claim 1 , further comprising at least one mutation corresponding to mutations in SEQ ID NO:1 selected from the group consisting of Y639F, S641A, F644Y, F667Y, E222K, S430P, F849I, F880Y, S633P, P266L, N748D, N748Q, Q758C and R756M.

8. The polymerase of claim 7 , comprising mutations corresponding to Y639F and S641A in SEQ ID NO:1.

9. The polymerase of claim 1 having greater resistance to 30 mM NaCl, 7.5 mM phosphate, or 20 μg/ml single stranded DNA than a wild-type T7 RNA polymerase having the sequence of SEQ ID NO:1 or a wild-type T3 RNA polymerase having the sequence of SEQ ID NO:3.

10. A method of amplifying mRNA comprising
(a) combining the mRNA with a reverse transcriptase and an appropriate first buffer and first reagents to form a first mixture and incubate the first mixture under conditions and for a time sufficient to synthesize a first strand of a cDNA;
(b) forming a second mixture by adding (i) DNA polymerase or an RNA polymerase having DNA polymerase activity and (ii) an appropriate second buffer and second reagents to the first mixture comprising the first strand cDNA, and incubating the second mixture under conditions and for a time sufficient to synthesize a second strand of the cDNA and form a double stranded cDNA (ds-cDNA); and
(c) forming a third mixture by adding an appropriate third buffer, third reagents and the polymerase of any one of claims 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 and 9 to the second mixture comprising the ds-cDNA, and incubating the third mixture under conditions and for a time sufficient to synthesize a needed amount of amplified RNA.

11. A composition comprising the polymerase of any one of claims 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 and 9 , and a reagent selected from a salt, phosphate, pyrophosphate or single stranded DNA at a concentration that is inhibitory to wild-type T7 RNA polymerase.

12. The composition of claim 11 , wherein said composition is in a kit, and the kit further comprises one or more reagents, buffers and/or enzymes for amplifying mRNA.

Assignees

• Coleman Jack
• Enzo Life Sciences Inc

Inventors

• Coleman Jack

Classifications

• C12N9/1247 Primary

  DNA-directed RNA polymerase (2.7.7.6) · CPC title