Thermostable DNA polymerase from Thermus thermophilus B35: preparation and study of a modified form of the enzyme with high affinity to ddNTP

A G Akishev; N I Rechkunova; J E Tomilova; N A Lebedeva; O I Lavrik; S Kh Degtyarev

doi:10.1023/b:biry.0000011651.66752.80

Thermostable DNA polymerase from Thermus thermophilus B35: preparation and study of a modified form of the enzyme with high affinity to ddNTP

Biochemistry (Mosc). 2003 Dec;68(12):1307-12. doi: 10.1023/b:biry.0000011651.66752.80.

Authors

A G Akishev¹, N I Rechkunova, J E Tomilova, N A Lebedeva, O I Lavrik, S Kh Degtyarev

Affiliation

¹ Institute of Molecular Biology and Biophysics, Siberian Branch of the Russian Academy of Medical Sciences, Novosibirsk 630117, Russia.

PMID: 14756626
DOI: 10.1023/b:biry.0000011651.66752.80

Abstract

The hybrid protein consisting of Tte DNA polymerase fragment and mutant Taq DNA polymerase (F667Y) fragment in the ratio 20 : 1 was constructed. Affinity of the modified enzyme (substitutions F669Y, V667I, and S692Q) to ddNTP was two orders higher than that of the wild type enzyme. The modified enzyme was used for sequencing DNA fragment with total deoxyguanosine and deoxycytidine content of 68%. In the polymerase chain reaction, the modified enzyme exhibits properties typical of the wild type Tte DNA polymerase.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Autoradiography
Base Sequence
Catalysis
DNA Primers / genetics
DNA Primers / metabolism
DNA-Directed DNA Polymerase / chemistry*
DNA-Directed DNA Polymerase / genetics
DNA-Directed DNA Polymerase / metabolism*
Enzyme Stability
Escherichia coli
Molecular Sequence Data
Mutation / genetics
Nucleosides / chemistry
Nucleosides / metabolism*
Sequence Alignment
Temperature*
Thermus thermophilus / enzymology*
Thermus thermophilus / genetics

Substances

DNA Primers
Nucleosides
DNA polymerase, Thermus thermophilus
DNA-Directed DNA Polymerase