Overproduction of Thermus aquaticus dna polymerase and its structural analysis by ion-spray mass spectrometry

Yoshizumi Ishino, Takashi Ueno, Masaru Miyagi, Takashi Uemori, Mitsuo Imamura, Susumu Tsunasawa, Ikunoshin Kato

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

We cloned the pol gene from the Thermus aquaticus YT-1 strain into a plasmid vector and constructed a high-level expression system of the gene in Escherichia coli. Six codons in the translational start region were changed to simple AT-type codons or codons which are most frequently used in E. coli by the genetic engineering techniques with retention of the amino acid sequence of the native enzyme. The modified pol genes were expressed under the lac promoter of pUC-type plasmid and 266, 418 units of activity was obtained in a sonicated and heated crude extract from 2 g of E. coli cells bearing one of the recombinant plasmids, pTAQ9. Highly purified protein was subjected to structural analysis using a protein sequencer and an ion-spray mass spectrometer combined with reversed-phase HPLC (LC-MS). The primary structure of the DNA polymerase was identical with the amino acid sequence deduced from the nucleotide sequence of the pol gene as far as examined (about 95% of the sequence); though, several regions where small peptides of less than 5 residues were produced by lysyl endopeptidase digestion could not be sequenced.

Original languageEnglish
Pages (from-to)1019-1024
Number of pages6
JournalJournal of biochemistry
Volume116
Issue number5
DOIs
Publication statusPublished - Jan 1 1994

Fingerprint

Taq Polymerase
pol Genes
Structural analysis
Codon
Mass spectrometry
Mass Spectrometry
Plasmids
lysyl endopeptidase
Genes
Ions
Escherichia coli
Amino Acid Sequence
Thermus
Genetic Techniques
Bearings (structural)
Genetic Engineering
DNA-Directed DNA Polymerase
Complex Mixtures
Amino Acids
Genetic engineering

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

Overproduction of Thermus aquaticus dna polymerase and its structural analysis by ion-spray mass spectrometry. / Ishino, Yoshizumi; Ueno, Takashi; Miyagi, Masaru; Uemori, Takashi; Imamura, Mitsuo; Tsunasawa, Susumu; Kato, Ikunoshin.

In: Journal of biochemistry, Vol. 116, No. 5, 01.01.1994, p. 1019-1024.

Research output: Contribution to journalArticle

Ishino, Yoshizumi ; Ueno, Takashi ; Miyagi, Masaru ; Uemori, Takashi ; Imamura, Mitsuo ; Tsunasawa, Susumu ; Kato, Ikunoshin. / Overproduction of Thermus aquaticus dna polymerase and its structural analysis by ion-spray mass spectrometry. In: Journal of biochemistry. 1994 ; Vol. 116, No. 5. pp. 1019-1024.
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