Overproduction of Thermus aquaticus DNA polymerase and its structural analysis by ion-spray mass spectrometry.
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.
Ishino
Y
Y
Biotechnology Research Laboratories, Takara Shuzo Co., Ltd., Shiga.
Ueno
T
T
Miyagi
M
M
Uemori
T
T
Imamura
M
M
Tsunasawa
S
S
Kato
I
I
D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics*; D004795:Enzyme Stability; D015870:Gene Expression; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017421:Sequence Analysis; D018629:Spectrometry, Mass, Secondary Ion; D013824:Thermus / Q000201:enzymology*
D004259:DNA-Directed DNA Polymerase