Molecular cloning and nucleotide sequence of the aminopeptidase T gene of Thermus aquaticus YT-1 and its high-level expression in Escherichia coli.
Aminopeptidase T (AP-T) is a metallo-dependent dimeric enzyme of Thermus aquaticus YT-1, an extremely thermophilic bacterium. We cloned the AP-T gene from T. aquaticus YT-1 into Escherichia coli using a synthetic oligonucleotide as a hybridization probe. The nucleotide sequence of the AP-T gene was found to encode 408 amino acid residues with GTG as a start codon. The molecular weight was calculated to be 44,820. The AP-T was overproduced in E. coli (about 5% of total soluble protein) when the start codon of the gene was changed from GTG to ATG, and the gene was downstream from the tac promoter. The AP-T expressed in E. coli was heat stable and easily purified by heat treatment (80 degrees C, 30 min). The N-terminal amino acid sequence of AP-T showed similarity with that of aminopeptidase II from Bacillus stearothermophilus.
Department of Agricultural Chemistry, University of Tokyo, Japan.
H
H
Motoshima
N
N
Azuma
S
S
Kaminogawa
M
M
Ono
E
E
Minagawa
H
H
Matsuzawa
T
T
Ohta
K
K
Yamauchi
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry / Q000235:genetics*; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D015247:Deoxyribonuclease HindIII; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006358:Hot Temperature; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015345:Oligonucleotide Probes; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013824:Thermus / Q000201:enzymology / Q000235:genetics*
D001426:Bacterial Proteins; D003062:Codon; D015345:Oligonucleotide Probes; D015247:Deoxyribonuclease HindIII; D000626:Aminopeptidases; C067298:aminopeptidase T protein, Thermus aquaticus