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.