Summary
We have cloned and determined the nucleotide sequence of a cDNA fragment for the entire coding region of the alkaline protease (Alp) from a filamentous ascomycete Aspergillus oryzae. According to the deduced amino acid sequence, Alp has a putative prepro region of 121 amino acids preceding the mature region, which consists of 282 amino acids. A consensus sequence of a signal peptide consiting of 21 amino acids is found at the N-terminus of the prepro region. The primary structure of the mature region shares extensive homology (29%–44%) with those of subtilisin families, and the three residues (Asp 32, His 64 and Ser 221 in subtilisin BPN′) composing the active site are preserved. The entire cDNA, coding for prepro Alp, when introduced into the yeast Saccharomyces cerevisiae, directed the secretion of enzymatically active Alp into the culture medium, with its N-terminus and specific activity identical to native Aspergillus Alp.
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References
Ballou CE (1982) Yeast cell wall and cell surface. In: Strathern J, Jones E, Broach J (eds) Molecular biology of the yeast Saccharomyces, vol 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp 335–360
Beggs JD (1978) Transformation of yeast by a replicating hybrid plasmid. Nature 275:104–109
Bussey H (1988) Proteases and the processing of precursors to secreted proteins in yeast. Yeast 4:17–26
Davidow LS, O'Donnell MM, Kaczmarek FS, Pereira DA, DeZeeuw JR, Franke AE (1987) Cloning and sequencing of the alkaline extracellular protease gene of Yarrowia lipolytica. J Bacteriol 169:4621–4629
Elliott SD, Liu T-Y (1970) Streptococcal proteinase. Methods Enzymol 19:252–261
Foltmann B (1970) Prochymosin and chymosin (prorennin and rennin). Methods Enzymol 19:421–436
Fukushima Y, Itch H, Fukase T, Motai H (1989) Continuous protease production in a carbon limited chemostat culture by salt tolerant Aspergillus oryzae. Appl Microbiol Biotechnol 30:604–608
Hanahan D (1985) Techniques for transformation of E. coli. In: Glover DM (ed) DNA cloning, vol 1. IRL Press, Oxford, England, pp 109–135
Hayashi K, Fukushima D, Mogi K (1967) Isolation of alkaline proteinase from Aspergillus sojae in homogeneous form. Agric Biol Chem 31:1237–1241
Innis MA, Holland MJ, McCabe PC, Cole GE, Wittman VP, Tal R, Watt KWK, Gelfand DH, Holland JP, Meade JH (1985) Expression, glycosylation, and secretion of an Aspergillus glucoamylase by Saccharomyces cerevisiae. Science 228:21–26
Jany K-D, Lederer G, Mayer B (1986) Amino acid sequence of proteinase K from the mold Tritirachium album Limber. FEBS Lett 199:139–144
Klionsky DJ, Banta LM, Emr SD (1988) Intracellular sorting signal and processing of a yeast vacuolar hydrolase: proteinase A propeptide contains vacuolar targeting information. Mol Cell Biol 5:2105–2116
Kozak M (1984) Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucleic Acids Res 12:857–872
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York
Meloun B, Baudys M, Kostka V, Hausdorf G, Frömmel C, Höhne WE (1985) Complete primary structure of thermitase from Thermoactinomyces vulgaris and its structural features related to the subtilisin-type proteinases. FEBS Lett 183:195–200
Moehle CM, Tizard R, Lemmon SK, Smart J, Jones EW (1987) Protease B of the lysosomelike vacuole of the yeast Saccharomyces cerevisiae is homologous to the subtilisin family of serine proteases. Mol Cell Biol 7:4390–4399
Nakagawa Y (1970) Alkaline proteinases from Aspergillus. Methods Enzymol 19:581–591
Nunberg JH, Meade JH, Cole G, Lawyer FC, McCabe P, Schweickart V, Tal R, Wittman VP, Flatgaard JE, Innis MA (1984) Molecular cloning and characterization of the glucoamylase gene of Aspergillus awamori. Mol Cell Biol 4:2306–2315
Pantoliano MW, Ladner RC, Bryan PN, Rollence ML, Wood JF, Poulos TL (1987) Protein engineering of subtilisin BPN': enhanced stabilization through the introduction of two cysteines to form a disulfide bond. Biochemistry 26:2077–2082
Perlman D, Halvorson HO (1983) A putative signal peptidase recognition site and sequence in eukaryotic and prokaryotic signal peptides. J Mol Biol 167:391–409
Power SD, Adams RM, Wells JA (1986) Secretion and autoproteolytic maturation of subtilisin. Proc Natl Acad Sci USA 83:3096–3100
Sato T, Tsunasawa S, Nakamura Y, Emi M, Sakiyama F, Matsubara K (1986) Expression of the human salivary α-amylase gene in yeast and characterization of the secreted protein. Gene 50:247–257
Tatsumi H, Ohsawa M, Tsuji RF, Murakami S, Nakano E, Motai H, Masaki A, Ishida Y, Murakami K, Kawabe H, Arimura H (1988a) Cloning and sequencing of the alkaline protease cDNA from Aspergillus oryzae. Agric Biol Chem 52:1887–1888
Tatsumi H, Masuda T, Nakano E (1988b) Synthesis of enzymatically active firefly luciferase in yeast. Agric Biol Chem 52:1123–1127
Valls LA, Hunter CP, Rothman JH, Stevens TH (1987) Protein sorting in yeast: the localization determinant of yeast vacuolar carboxypeptidase Y resides in the propeptide. Cell 48:887–897
Wells JA, Ferrari E, Henner DJ, Estell DA, Chen EY (1983) Cloning, sequencing, and secretion of Bacillus amyloliquefaciens subtilisin in Bacillus subtilis. Nucleic Acids Res 11:7911–7925
Wilcox PE (1970) Chymotrypsinogens-chymotrypsins. Methods Enzymol 19:64–108
Wright CS, Alden RA, Kraut J (1969) Structure of subtilisin BPN' at 2.5 Å resolution. Nature 221:235–242
Yamashita T, Tonouchi N, Uozumi T, Beppu T (1987) Secretion of Mucor rennin, a fungal aspartic protease of Mucor pusillus, by recombinant yeast cells. Mol Gen Genet 210:462–467
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Communicated by C.P. Hollenberg
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Tatsumi, H., Ogawa, Y., Murakami, S. et al. A full length cDNA clone for the alkaline protease from Aspergillus oryzae: Structural analysis and expression in Saccharomyces cerevisiae . Molec. Gen. Genet. 219, 33–38 (1989). https://doi.org/10.1007/BF00261154
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DOI: https://doi.org/10.1007/BF00261154