Recombinant his-tagged DNA polymerase. I. Cloning, purification and partial characterization of Thermus thermophilus recombinant DNA polymerase

被引：7

作者：

Dabrowski, S ^{[1
]}

Kur, J ^{[1
]}

机构：

[1] Gdansk Univ Technol, Dept Microbiol, PL-80952 Gdansk, Poland

来源：

ACTA BIOCHIMICA POLONICA | 1998年 / 45卷 / 03期

关键词：

PCR; RT-PCR; DNA polymerase; reverse transcriptase; Thermus thermophilus; recombinant protein;

D O I：

10.18388/abp.1998_4258

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

The Tth DNA polymerase gene from the thermophilic Thermus thermophilus (strain HB8) was amplified, cloned and expressed in Escherichia coli. The recombinant DNA polymerase containing a polyhistidine tag at the N-terminus was isolated in a single step by Ni2+ affinity chromatography. The purified recombinant enzyme, showing high polymerase activity contained 43 additional amino-acid residues (including a cluster of six histidine residues inserted for purification of the recombinant protein by metal-affinity chromatography) at N-terminus. The applied overexpression system was very efficient giving 700000 u of DNA polymerase activity from 1 liter of induced culture. The enzyme was characterized and displayed high DNA polymerase and reverse transcriptase activities and high thermostability as compared to the native Tth DNA polymerase.

引用

页码：653 / 660

页数：8

共 21 条

[1]

BUSH GL, 1991, J BIOL CHEM, V266, P13811

[2] DEOXYRIBONUCLEIC-ACID POLYMERASE FROM EXTREME THERMOPHILE THERMUS-AQUATICUS [J].

CHIEN, A ;

EDGAR, DB ;

TRELA, JM .

JOURNAL OF BACTERIOLOGY, 1976, 127 (03) :1550-1557

[3] THERMOSTABLE DNA-POLYMERASE FROM THE ARCHAEBACTERIUM SULFOLOBUS-ACIDOCALDARIUS - PURIFICATION, CHARACTERIZATION AND IMMUNOLOGICAL PROPERTIES [J].

ELIE, C ;

DERECONDO, AM ;

FORTERRE, P .

EUROPEAN JOURNAL OF BIOCHEMISTRY, 1989, 178 (03) :619-626

[4]

Hochuli E, 1990, Genet Eng (N Y), V12, P87

[5] GENETIC APPROACH TO FACILITATE PURIFICATION OF RECOMBINANT PROTEINS WITH A NOVEL METAL CHELATE ADSORBENT [J].

HOCHULI, E ;

BANNWARTH, W ;

DOBELI, H ;

GENTZ, R ;

STUBER, D .

BIO-TECHNOLOGY, 1988, 6 (11) :1321-1325

[6]

INNIS MA, 1990, PCR PROTOCOLS GUIDE, P146

[7] COMPILATION AND ALIGNMENT OF DNA-POLYMERASE SEQUENCES [J].

ITO, J ;

BRAITHWAITE, DK .

NUCLEIC ACIDS RESEARCH, 1991, 19 (15) :4045-4057

[8] RAPID AND EFFICIENT PURIFICATION OF NATIVE HISTIDINE-TAGGED PROTEIN EXPRESSED BY RECOMBINANT VACCINIA VIRUS [J].

JANKNECHT, R ;

DEMARTYNOFF, G ;

LOU, J ;

HIPSKIND, RA ;

NORDHEIM, A ;

STUNNENBERG, HG .

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1991, 88 (20) :8972-8976

[9]

KASLOW DC, 1994, BIO-TECHNOL, V12, P494, DOI 10.1038/nbt0594-494

[10] PURIFICATION AND CHARACTERIZATION OF DNA-POLYMERASE FROM THE ARCHAEBACTERIUM SULFOLOBUS-ACIDOCALDARIUS [J].

KLIMCZAK, LJ ;

GRUMMT, F ;

BURGER, KJ .

NUCLEIC ACIDS RESEARCH, 1985, 13 (14) :5269-5282

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