Open AccessRecombinant His-tagged DNA polymerase. II. Cloning and purification of Thermus aquaticus recombinant DNA polymerase (Stoffel fragment).
Author(s) -
Sławomir Dąbrowski,
Józef Kur
Publication year - 1998
Publication title -
acta biochimica polonica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.452
H-Index - 78
eISSN - 1734-154X
pISSN - 0001-527X
DOI - 10.18388/abp.1998_4204
Subject(s) - thermus aquaticus , recombinant dna , microbiology and biotechnology , thermus , taq polymerase , dna polymerase , dna polymerase i , polymerase , molecular cloning , biology , hot start pcr , escherichia coli , dna , thermostability , polymerase chain reaction , biochemistry , chemistry , enzyme , thermophile , peptide sequence , gene , nested polymerase chain reaction , reverse transcriptase
The Stoffel DNA fragment, shortened by 12 bp from 5' end, coding for Stoffel DNA polymerase (missing 4 amino acids at N-terminus of Stoffel amino-acids sequence) from the thermophilic Thermus aquaticus (strain YT-1) was amplified, cloned and expressed in Escherichia coli. The recombinant Stoffel fragment contained a polyhistidine tag at the N-terminus (21 additional amino acids) that allowed its single-step isolation by Ni2+ affinity chromatography. The enzyme was characterized and displayed high DNA polymerase activity and thermostability evidently higher than the native Taq DNA polymerase.