Molecular characterization of a novel subtilisin inhibitor protein produced by Streptomyces venezuelae CBS762.70.
We report here on the isolation and identification of a gene coding for a novel subtilisin inhibitor (VSI) isolated from Streptomyces venezuelae CBS762.70. The vsi gene was isolated on a 5-kb chromosomal PvuII fragment as identified by DNA sequencing and inhibitor activity testing of the gene product. Primer extension studies revealed that the mRNA transcriptional start point was situated at -37 and -36 relatively to the ATG start codon assuming the presence of solely one promoter. Vsi promoter strength was about double of those of ermE-P1a and aph-P1, as tested with the mRNA production of the aphII gene preceded by the respective promoters. Translation of the vsi coding sequence revealed a 28 amino acids long signal peptide. The mature VSI protein consists of 118 amino acids of which 87% was verified by N-terminal amino acid sequence analysis. Compared with the already known Streptomyces proteinase inhibitors, VSI shows a relatively high amino acid identity in the conserved domains. Nevertheless, only a maximum amino acid identity of 56.1% was noticed and some highly conserved residues were substituted in VSI. As a consequence, VSI could be classified within a separate group of Streptomyces subtilisin inhibitors.
Laboratorie of Microbiology, Rega Institute, K.U. Leuven, Belgium.
L
L
Van Mellaert
E
E
Lammertyn
S
S
Schacht
P
P
Proost
J
J
Van Damme
B
B
Wroblowski
J
J
Anné
T
T
Scarcez
E
E
Sablon
J
J
Raeymaeckers
A
A
Van Broekhoven
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000302:isolation & purification; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011480:Protease Inhibitors / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism; D013381:Subtilisins / Q000037:antagonists & inhibitors* / Q000378:metabolism
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D011480:Protease Inhibitors; C116044:subtilisin inhibitor protein VSI, Streptomyces venezuelae; D013381:Subtilisins