Cloning and characterization of a gene encoding a secreted tripeptidyl aminopeptidase from Streptomyces lividans 66.
The gene encoding a tripeptidyl aminopeptidase (Tap) from Streptomyces lividans was cloned by using a simple agar plate activity assay. Overexpression of the cloned gene results in the production of a secreted protein which has an apparent subunit molecular weight of 55,000 and is responsible for the major amino-terminal degradative activity in culture broths of S. lividans strains. A DNA sequence analysis revealed a potential protein-encoding region of the size expected to encode the observed protein, which contained a sequence that exhibited significant homology around a putative active site serine residue observed for lipases, esterases, and acyl transferases. Preceding the amino terminus of the secreted protein was a predicted signal peptide of 36 amino acids followed by a tripeptide, which could be autocatalytically removed from a secreted Tap precursor. The transcriptional start site for the gene was mapped by primer extension. Mutant strains of S. lividans lacking detectable Tap activity were able to grow and sporulate normally. Cross-species hybridization experiments showed that DNA homologs of the tap gene are present in most of the Streptomyces strains tested.
D000595:Amino Acid Sequence; D000626:Aminopeptidases; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D010450:Endopeptidases / Q000235:genetics*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D015183:Restriction Mapping; D013045:Species Specificity; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics*
D017931:DNA Primers; D004269:DNA, Bacterial; D021382:Protein Sorting Signals; D010450:Endopeptidases; D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases