# US 9,040,276 B2 · DNA binding protein-polymerase chimeras

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- Number: US 9,040,276 B2
- Document ID: US9040276B2
- Status: Granted
- Kind: B2
- Application number: 11/488,535
- Filed: 2006-07-17
- Published: 2007-06-21
- Granted: 2015-05-26
- Earliest claimed: 2005-07-15
- Assignees: Agilent Technologies, Inc.
- Applicants: Michael Borns
- Inventors: Michael Borns
- Web page: https://patentgrep.com/patent/US9040276B2

## CPC

- C12N 9/1252: {DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase}
- C07K 2319/80: containing a DNA binding domain, e.g. Lacl or Tet-repressor
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

The invention relates to compositions and methods directed to chimeric DNA polymerases, which comprise a mutated DNA binding polypeptide domain and a mutated or wild-type DNA polymerase polypeptide domain.

## Claims

### Claim 1

A chimeric DNA polymerase comprising a DNA binding domain and a polymerase domain, wherein said DNA binding domain has the wild-type sequence of Sso7d (SEQ ID NO:2) or of a wild-type Sso7d-like protein selected from the group consisting of Ssh7b (SEQ ID NO:8), RiboP3 (SEQ ID NO:9), Stole (SEQ ID NO:10), Sac7d (SEQ ID NO:12), Sac7e (SEQ ID NO:14), and Sac7a (SEQ ID NO:16), with the exception of seven, eight, or nine substitution mutations at positions corresponding to seven, eight, or all nine of the following amino acid positions: 13, 16, 40, 41, 45, 55, 56, 61, and 63 of SEQ ID NO:2.

### Claim 2 (depends on 1)

The chimeric DNA polymerase of claim 1, wherein said substitution mutations are selected from the group consisting of:
an E or Q the position corresponding to position 13 of SEQ ID NO:2;
an I or T at the position corresponding to position 16 of SEQ ID NO:2;
a T or S at the position corresponding to position 40 of SEQ ID NO:2;
a G or A at the position corresponding to position 41 of SEQ ID NO:2;
a V or L at the position corresponding to position 45 of SEQ ID NO:2;
an L or V at the position corresponding to position 55 of SEQ ID NO:2;
a Q or D at the position corresponding to position 56 of SEQ ID NO:2;
a Q or E at the position corresponding to position 61 of SEQ ID NO:2; and
a K or R at the position corresponding to position 63 of SEQ ID NO:2.

### Claim 3

A chimeric DNA polymerase comprising a DNA binding domain and a polymerase domain, wherein said DNA binding domain has the sequence of Sso7d (SEQ ID NO:2) or of a wild-type Sso7d-like protein selected from the group consisting of Ssh7b (SEQ ID NO:8), RiboP3 (SEQ ID NO:9), Stole (SEQ ID NO:10), Sac7d (SEQ ID NO:12), Sac7e (SEQ ID NO:14), and Sac7a (SEQ ID NO:16), with the exception of seven substitution mutations at the positions corresponding to amino acid positions 13, 16, 40, 45, 55, 56, and 63 of SEQ ID NO:2.

### Claim 4 (depends on 3)

The chimeric DNA polymerase of claim 3, wherein said substitution mutations are selected from the group consisting of:
an E or Q the position corresponding to position 13 of SEQ ID NO:2;
an I or T at the position corresponding to position 16 of SEQ ID NO:2;
a T or S at the position corresponding to position 40 of SEQ ID NO:2;
a V or L at the position corresponding to position 45 of SEQ ID NO:2;
an L or V at the position corresponding to position 55 of SEQ ID NO:2;
a Q or D at the position corresponding to position 56 of SEQ ID NO:2;
a K or R at the position corresponding to position 63 of SEQ ID NO:2.

### Claim 5

A chimeric DNA polymerase comprising a DNA binding domain and a polymerase domain, wherein said DNA binding domain comprises the amino acid sequence of SEQ ID NO:4.

### Claim 6

A chimeric DNA polymerase comprising a DNA binding domain and a polymerase domain, wherein said DNA binding domain has the sequence of Sso7d (SEQ ID NO:2) or of a wild-type Sso7d-like protein selected from the group consisting of Ssh7b (SEQ ID NO:8), RiboP3 (SEQ ID NO:9), Stole (SEQ ID NO:10), Sac7d (SEQ ID NO:12), Sac7e (SEQ ID NO:14), and Sac7a (SEQ ID NO:16), with the exception of nine substitution mutations at the positions corresponding to amino acid positions 13, 16, 40, 41, 45, 55, 56, 61 and 63 of SEQ ID NO:2.

### Claim 7 (depends on 1)

The chimeric DNA polymerase of claim 1, wherein said polymerase domain comprises an archaeal DNA polymerase.

### Claim 8 (depends on 1)

The chimeric DNA polymerase of claim 1, wherein said polymerase domain has thermally stable polymerase activity.

### Claim 9 (depends on 1)

The chimeric DNA polymerase of claim 1, wherein said polymerase domain comprises a Pfu DNA polymerase.

### Claim 10 (depends on 9)

The chimeric DNA polymerase of claim 9, wherein said polymerase domain is at least 95% identical with the amino acid sequence of SEQ ID NO:18.

### Claim 11 (depends on 1)

The chimeric DNA polymerase of claim 1, wherein said polymerase domain comprises a Taq DNA polymerase.

### Claim 12

A chimeric DNA polymerase comprising the amino acid sequence of SEQ ID NO:20.

### Claim 13 (depends on 1)

A composition comprising the DNA polymerase of claim 1, or 12.

### Claim 14 (depends on 1)

A kit comprising the chimeric DNA polymerase of claim 1, or 12, and packaging materials therefor.

### Claim 15 (depends on 1)

A method for DNA synthesis comprising:
a) providing a chimeric DNA polymerase according to claim 1, or 12; and
b) contacting said chimeric DNA polymerase with a nucleic acid template, wherein said enzyme permits DNA synthesis.

### Claim 16 (depends on 1)

The chimeric DNA polymerase of claim 1, wherein said polymerase domain comprises a mutant polymerase.

### Claim 17 (depends on 16)

The chimeric DNA polymerase of claim 16, wherein said mutant polymerase has V93R, V93E, V93D, V93K or V93N mutations resulting in reduced uracil detection activity.

### Claim 18 (depends on 16)

The chimeric DNA polymerase of claim 16, wherein said mutant polymerase has D141A and/or E143A mutations resulting in reduced 3′-5′ exonuclease activity.

### Claim 19 (depends on 6)

The chimeric DNA polymerase of claim 6, wherein said chimeric DNA polymerase has
an E or Q the position corresponding to position 13 of SEQ ID NO:2;
an I or T at the position corresponding to position 16 of SEQ ID NO:2;
a T or S at the position corresponding to position 40 of SEQ ID NO:2;
a G or A at the position corresponding to position 41 of SEQ ID NO:2;
a V or L at the position corresponding to position 45 of SEQ ID NO:2;
an L or V at the position corresponding to position 55 of SEQ ID NO:2;
a Q or D at the position corresponding to position 56 of SEQ ID NO:2;
a Q or E at the position corresponding to position 61 of SEQ ID NO:2; and
a K or R at the position corresponding to position 63 of SEQ ID NO:2.