Cloning and expression in yeast of a cDNA clone encoding Aspergillus oryzae neutral protease II, a unique metalloprotease.
The neutral protease II (NpII) from Aspergillus oryzae is a zinc-containing metalloprotease with some unique properties. To elucidate its structure, we isolated a full-length cDNA clone for NpII. Sequence analysis reveals that NpII has a prepro region consisting of 175 amino acids preceding the mature region, which consists of 177 amino acids. As compared with other microbial metalloproteases, NpII is found to be unique in that it shares only a limited homology with them around two zinc ligand His residues and that the positions of the other zinc ligand (Glu) and the active site (His) cannot be established by homology. When a plasmid designed to express the prepro NpII cDNA was introduced into Saccharomyces cerevisiae and the transformant was cultured in YPD medium (2% glucose, 2% polypeptone, 1% yeast extract), it secreted a proNpII. However, in a culture of the same medium containing 0.2 mM ZnCl2, it secreted a mature NpII with a specific activity and N-terminus identical to those of native NpII. This observation suggests that either an autoproteolytic activity or a yeast protease effected the processing.
Tatsumi
H
H
Research and Development Division, Kikkoman Corporation, Chiba, Japan.
Murakami
S
S
Tsuji
R F
RF
Ishida
Y
Y
Murakami
K
K
Masaki
A
A
Kawabe
H
H
Arimura
H
H
Nakano
E
E
Motai
H
H
D000595:Amino Acid Sequence; D001236:Aspergillus oryzae / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D015870:Gene Expression; D005800:Genes, Fungal; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D014170:Transformation, Genetic
D008666:Metalloendopeptidases; C070788:neutral protease II, Aspergillus oryzae