# US 5,352,778 A · Recombinant thermostable DNA polymerase from archaebacteria

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- Number: US 5,352,778 A
- Document ID: US5352778A
- Status: Granted
- Kind: A
- Application number: 08/167,238
- Filed: 1993-12-15
- Granted: 1994-10-04
- Earliest claimed: 1990-04-26
- Assignees: New England Biolabs, Inc.
- Inventors: Donald G. Comb; Francine Perler; Rebecca Kucera; William E. Jack
- Web page: https://patentgrep.com/patent/US5352778A

## CPC

- C07K 16/12: against material from bacteria
- C12N 9/1252: {DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase}
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Recombinant DNA polymeraset from archaebacteria as well as isolated DNA coding for such polymeraset are provided. The isolated DNA is obtained by use of DNA or antibody probet prepared from the DNA encoding T. litoralis DNA polymerate and the T. litoralis DNA polymerate respectively. Also provided are meshocs for producing recombinant archaebacteria thermostable DNA polymerase and methods for enhancing the expression of such polymeraset by identifying, locating and removing introns from within the DNA coding for such DNA polymerases.

## Claims

### Claim 1

An isolated DNA which codes for a recombinant DNA polymerase from thermophilic archaebacteria, consisting essentially of a DNA fragment which hybridizes in a Southern blot to an isolated DNA fragment selected from the group consisting of a DNA fragment consisting essentially of nucleotides 1-1274 of SEQ ID NO:1, a DNA fragment consisting essentially of nucleotides 291-1772 of SEQ ID NO: 1, a DNA fragment consisting essentially of nucleotides 3387-3533 of SEQ ID NO:1, a DNA fragment consisting essentially of nucleotides 4704-5396 of SEQ ID NO: 1, and a DNA fragment consisting essentially of nucleotides 4718-5437 of SEQ ID NO:1, wherein hybridization is conduction under the following conditions:
a) hybridization: 0.75M NaCI, 0.15 Tris, 10mM EDTA, 0.1% Sodium Pyrophosphate, 0.1% SLS, 0.03% BSA, 0.03% Ficoll 400, 0.03% PVP and 100ug/ml boiled calf thymus DNA at 50.degree. C. for about 12 hours and;
b) wash: 3.times.30 minutes with 0.1X SET, 0.1% SDS, 0.1% sodium pryrophosphate and 0.1M phosphate buffer at 45.degree. C.

### Claim 2 (depends on 1)

A cloning vector comprising the isolated DNA of claim 1.

### Claim 3 (depends on 2)

A host cell transformed by the vector of claim 2.

### Claim 4 (depends on 2)

A method for producing a recombinant thermostable DNA polymerase from archaebacteria comprising culturing a host cell transformed with the vector of claim 2 under conditions suitable for the expression of the DNA polymerase.

### Claim 5 (depends on 1)

A DNA probe which hybridizes to the DNA sequence coding for the archaebacteria thermostable DNA polymerase of claim 1, wherein the DNA probe is selected from the group consisting of a DNA fragment consisting essentially of nucleotides 1-4771 of SEQ ID NO: 1, a DNA fragment consisting essentially of nucleotides 1-1274 of SEQ ID NO:1, a DNA fragment consisting essentially of nucleotides 1269-2856 of SEQ ID NO:1, and a DNA fragment consisting essentially of nucleotides 2851-4771 of SEQ ID NO: 1.

### Claim 6

A method for isolating a target DNA fragment consisting essentially of a DNA coding for a thermostable DNA polymerase from thermophilic archaebacterium comprising the steps of:
(a) forming a genomic library from the archaebacterium;
(b) transforming or transfecting an appropriate host cell with the library of step (a);
(c) contacting DNA from the transformed or transfected host cell with a DNA probe which hybridizes to a DNA fragment selected from the group consisting of a DNA fragment consisting essentially of nucleotides 1-1274 of SEQ ID NO:1, a DNA fragment consisting essentially of nucleotides 291-1772 of SEQ ID NO:1, a DNA fragment consisting essentially of nucleotides 3387-3533 of SEQ ID NO:1, a DNA fragment consisting essentially of nucleotides 4704-5396 of SEQ ID NO: 1, and a DNA fragment consisting essentially of nucleotides 4718-5437 of SEQ ID NO:1, wherein hybridization is conducted under the following conditions:
a) hybridization: 0.75M NaCI, 0.15 Tris, 10mM EDTA, 0.1% Sodium Pyrophosphate, 0.1% SLS, 0.03% BSA, 0.03% Ficoll 400, 0.03% PVP and 100ug/ml boiled calf thymus DNA at 50.degree. C. for about 12 hours and;
b) wash: 3.times.30 minutes with 0.1X SET, 0. 1% SDS, 0.1% sodium pryrophosphate and 0.1M phosphate buffer at 37.degree.-55.degree. C.;
(d) assaying the transformed or transfected cell of step (c) which hybridizes to the DNA probe for DNA polymerase activity;
(e) isolating a target DNA fragment which codes for the thermostable DNA polymerase.

### Claim 7

A method for isolating a DNA fragment consisting essentially of a DNA coding for a thermostable DNA polymerase from thermophilic archaebacterium comprising the steps of:
(a) forming a genomic library from the archaebacterium;
(b) transforming or transfecting an appropriate host cell with the library of step (a);
(c) contacting cellular polypeptide extract from the transformed or transfected host cell with an antibody probe which has specific affinity for T. litoralis DNA polymerase;
(d) assaying the transformed or transfected cell of step (c) which is cross-reactive to the antibody probe for DNA polymerase activity; and
(e) isolating a DNA fragment which codes for the thermostable DNA polymerase.

### Claim 8 (depends on 1)

A method for increasing the recombinant, heterologous expression in a host cell of a thermostable DNA polymerase from a thermophilic archaebacteria comprising the steps of:
(a) identifying and locating an intervening nucleotide sequence of the isolated DNA of claim 1; and
(b) removing the intervening nucleotide sequence from the isolated DNA.

### Claim 9 (depends on 8)

The method of claim 8, wherein the archaebacteria comprises T. litoralis.

### Claim 10 (depends on 9)

The method of claim 9, wherein the intervening nucleotide sequence is selected from the group of IVS1, IVS2 or IVS1 and IVS2.

### Claim 11 (depends on 8)

The method of claim 8, wherein the intervening nucleotide sequence is identified and located with a DNA probe coding for an intron from the DNA sequence encoding T. litoralis DNA polymerase.

### Claim 12 (depends on 11)

The method of claim 11, wherein the DNA probe is selected from the group consisting of a DNA fragment consisting essentially of the 1614 bp nucleotide sequence of SEQ ID NO:1 from nucleotide 1776 to nucleotide 3389, a DNA fragment consisting essentially of a portion of the 1614 bp nucleotide sequence of SEQ ID NO:1 from nucleotide 1776 to nucleotide 3389, a DNA fragment consisting essentially of 1160 bp nucleotide sequence of SEQ ID NO: 1 from nucleotide 3544 to nucleotide 4703, and a DNA fragment consisting essentially of a portion of the 1160 bp nucleotide sequence of SEQ ID NO:1 from nucleotide 3544 to nucleotide 4703.