Recombinant thermostable DNA polymerase from archaebacteria

• US 5,352,778 A
• Filed: 12/15/1993
• Issued: 10/04/1994
• Est. Priority Date: 04/26/1990
• Status: Expired due to Term

• Current Assignee: New England Biolabs Incorporated
• Original Assignee: New England Biolabs Incorporated
• Inventors: Kucera, Rebecca, Jack, William E., Perler, Francine, Comb, Donald G.
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Moore, William W.
• Application Number:
  US08/167,238
• Time in Patent Office: 293 Days
• Field of Search: 435/69.1, 435/194, 435/252.3, 435/252.33, 435/320.1, 536/23.2, 536/24.32
• US Class Current: 536/23.2
• CPC Class Codes: C07K 16/12 against material from bacteria
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

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

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°

C. for about 12 hours and;

b) wash;

3×

30 minutes with 0.1X SET, 0.1% SDS, 0.1% sodium pryrophosphate and 0.1M phosphate buffer at 45°

C.

2. A cloning vector comprising the isolated DNA of claim 1.

3. A host cell transformed by the vector of claim 2.

4. 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.

5. 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.

8. 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.

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

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

11. 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.

12. 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.

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°

C. for about 12 hours and;

b) wash;

3×

30 minutes with 0.1X SET, 0. 1% SDS, 0.1% sodium pryrophosphate and 0.1M phosphate buffer at 37°

-55°

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.

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.