Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1994-11-18
1997-09-09
Wax, Robert A.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
435219, 435896, C12N 952, C12N 950, C12N 100, C12N 120
Patent
active
056655860
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to a novel protease for cleaving the carboxyl side of a glutamic acid residue in an amino acid sequence of a polypeptide and a DNA sequence for encoding the protease.
BACKGROUND OF THE INVENTION
As an enzyme for acting on a protein (polypeptide) so as to specifically cleave the carboxyl side of a glutamic acid (Glu) residue, V8 protease derived from Staphylococcus aureus V8 strain is known (Gabriel R. Drapeau et al., J. Biol. Chem. 247, 20, 6720-6726 (1972)). This enzyme is classified as a serine protease. C. Carmona et al. (Nucl. Acids Res., 15, 6757 (1987)) cloned a DNA sequence for encoding this enzyme.
A similar enzyme, acidic amino acid specific endopeptidase derived from Streptomyces griseus, which is an actinomyces, is also known (Norio Yoshida et al., J. Biochem. 104, 3, 451-456 (1988)). Recently, I. Svendsen et al. reported an amino acid sequence of the acidic amino acid specific endopeptidase purified from Streptomyces griseus (FEBS LETTERS 292 165-167 (1991)). Furthermore, glutamic acid specific endopeptidase derived from Bacillus subtilis is also known (Takuro Niidome et al., J. Biochem. 108, 965-970 (1990)). With respect to some of these proteases, the characteristics thereof have been studied and inhibitors against them are known (K. Nagata et al., J. Biochem. 110, 859-862 (1991) and T. Komiyama, J. Biol. Chem. 266, 17, 10727-10730 (1991)).
The aforementioned enzymes are useful for specifically cleaving a protein at the aforementioned site for the purpose of structural analysis of the protein; for obtaining an objective protein through cleavage of a fusion protein in the case where the objective protein is desired to be produced as the fusion protein by a genetic recombination technique and the like. In the latter case, for example, after producing the objective protein by coupling with another protein through a Glu residue, the resultant is subjected to cleavage with one of the enzymes. Thus, the objective protein can be separated. Accordingly, there is a demand for a protease having such an enzymatic activity other than the above-mentioned enzymes.
DISCLOSURE OF THE INVENTION
The objective of this invention is providing a novel protease having an enzymatic activity for cleaving the carboxyl side of a Glu residue, and a DNA sequence for encoding the protease.
The present inventors have made various studies to obtain a protease having the activity of an acidic amino acid specific endopeptidase (in particular, a glutamic acid specific endopeptidase) from a strain of a microorganism other than the above-mentioned microorganisms. As a result, they found a novel protease having the above-mentioned characteristic derived from Streptomyces fradiae, and studied the characteristics thereof in detail. Furthermore, they determined a DNA sequence for encoding this protease, thereby accomplishing the present invention.
The protease of this invention cleaves a peptide bond including a carboxyl group of a glutamic acid residue in a peptide and has the following characteristics:
In a preferred embodiment, the protease is derived from Streptomyces fradiae.
In a preferred embodiment, the protease is derived from Streptomyces fradiae ATCC 14544 strain.
In a preferred embodiment, the protease comprises an amino acid sequence from Val in the 1 position to Tyr in the 187 position of SEQ ID No. 1.
The DNA sequence of this invention encodes any one of the aforementioned proteases. In a preferred embodiment, the DNA sequence comprises a base sequence from G in the 945 position to C in the 1505 position of SEQ ID No. 1.
In a preferred embodiment, the DNA sequence encodes a protease comprising an amino acid sequence from Met in the -170 position to Tyr in the 187 position of SEQ ID No. 1.
In a preferred embodiment, the DNA sequence comprises a base sequence from A in the 435 position to C in the 1505 position of SEQ ID No. 1.
The protease of the invention (hereinafter referred to as the SFase) is produced from Streptomyces, particularly a strain belonging to
REFERENCES:
C. Carmona & G. Gray, "Nucleotide Sequence of the Serine Protease Gene of Staphylococus aureus, Strain V8", Nucleic Acids Res., 15, p. 6757 (1987).
G. Drapeau et al., "Purification and Properties of an Extracellular Protease of Staphylococus aureus", J. Biol. Chem., 247, pp. 6720-6726 (1972).
T. Komiyama et al., "Replacement of P.sub.1 Leu.sup.18 by Glu.sup.18 in the Reactive Site of Turkey Ovomucoid Third Domain Converts It into a Strong Inhibitor of Glu-specific Streptomyces griseus Proteinase (GluSGP)", J. Biol. Chem., 266, pp. 10727-10730 (1991).
K. Nagata et al., "Subsite Mapping of an Acidic Amino Acid-Specific Endopeptidase from Streptomyces griseus, GluSGP, and Protease V8", J. Biochem., 110, pp. 859-862 (1991).
T. Niidome et al., "Purification and Characterization of an Acidic Amino Acid-Specific Endopeptidase of Bacillus subtilis Obtained from a Commercial Preparation (Protease Type XVI, Sigma)", J. Biochem., 108, pp. 965-970 (1990).
I. Svendsen & K. Breddam, "Isolation and Amino Acid Sequence of a Glutamic Acid Specific Endopeptidase from Bacillus licheniformis", Eur. J. Biochem., 204, pp. 165-171 (1992).
I. Svendsen et al., "The Primary Structure of the Glutamic Acid-Specific Protease of Streptomyces griseus", FEBS, 292, pp. 165-167 (1991).
N. Yoshida et al., "Purification and Characterization of an Acidic Amino Acid Specific Endopeptidase of Streptomyces griseus Obtained from a Commercial Preparation (Pronase)", J. Biochem., 104, pp. 451-456 (1988).
U. Sinha et al., "Two New Extracellular Serine Proteases From Streptomyces fradiae," Int. J. Biochem., 23, pp. 979-984 (1991).
K. Kitadokoro et al., "Purification, Characterization, and Molecular Cloning of an Acidic Amino Acid-Specific Proteinase From Streptomyces fradiae ATCC 14544," Biochim. Biophys. Acta, 1163, pp. 149-157 (1993).
Kitadokoro Kengo
Nakamura Etsuo
Shin Masaru
Teraoka Hiroshi
Tsuzuki Hiroshige
Haley, Jr. James F.
Hobbs Lisa J.
Shionogi & Co. Ltd.
Wax Robert A.
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