Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Patent
1995-06-01
1998-04-21
Wax, Robert A.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
435 691, 4351723, 435221, 435222, 4352523, 43525231, 4353201, 536 232, C12P 2100, C12N 954, C12N 956, C12N 1557
Patent
active
057416648
ABSTRACT:
The bacterial serine protease, subtilisin BPN', has been mutated so that it will efficiently and selectively cleave substrates containing dibasic residues. A combination mutant, where Asn 62 was changed to Asp and Gly 166 was changed to Asp (N62D/G166D), had a larger than additive shift in specificity toward dibasic substrates. Suitable substrates of the variant subtilisin were revealed by sorting a library of phage particles (substrate phage) containing five contiguous randomized residues. This method identified a particularly good substrate, Asn-Leu-Met-Arg-Lys-, that was selectively cleaved in the context of a fusion protein by the N62D/G166D subtilisin variant. Accordingly, this variant subtilisin may be useful for cleaving fusion proteins with dibasic substrate linkers and processing hormones or other proteins (in vitro or in vivo) that contain dibasic cleavage sites.
REFERENCES:
patent: 4760025 (1988-07-01), Estell et al.
patent: 5371008 (1994-12-01), Carter et al.
Bode et al., "Refined 1.2 A crystal structure of the complex formed between subtilisin Carlsberg and the inhibitor eglin c. Molecular structure of eglin and its detailed interaction with subtilisin" EMBO Journal 5(4):813-818 (1986).
Brenner et al., "Structural and enzymatic characterization of a purified prohormone-processing enzyme: Secreted, soluble Kex2 protease" Proc. Natl. Acad. Sci. USA 89:922-926 (1992).
Bresnahan, P. A. et al., "Human fur gene encodes a yeas kex2-like endoprotease that cleaves pro-.beta.-NGF in vivo" Journal of Cell Biology 111(6, Pt 2):2851-2859 (1990).
Carter et al., "Engineering Subtilisin BPN' for Site-Specific Proteolysis" Proteins: Struct. Funct., Genet. 6:240-248 (1989).
Creemers et al., "Modulation of Furin-Mediated Proprotein Processing Activity by Site-directed Mutagenesis" The Journal of Biological Chemistry 268(29):21826-21834 (1993).
Drenth et al., "Subtilisin Novo; The Three-Dimensional Structure and Its Comparison with Subtilisin BPN'" European Journal of Biochemistry 26:177-181 (1972).
Eder et al., "Hydrolysis of Small Peptide Substrates Parallels Binding of Chymotrypsin Inhibitor 2 for Mutants of Subtilisn BPN'" Federation of European Biochemical Societies 335(3):349-352 (1993).
Forsberg et al., "An Evaluation of Different Enzymatic Cleavage Methods for Recombination Fusion Proteins, Applied on Des(1-3) Insulin-Like Growth Factor I" Journal of Protein Chemistry 11(2):201-211 (1992).
Graf et al., "Electrostatic Complementarity Within the Substrate-Binding Pocket of Trypsin" Proc. Natl. Acad. Sci. 85:4961-4965 (1988).
Hedstrom et al., "Converting Trypsin to Chymotrypsin: The Role of Surface Loops" Science, 255(5049):1249-1253 (1992).
Hosaka et al., "Arg-X-Lys/Arg-Arg Motif as a Signal for Precursor Cleavage Catalyzed by Furin within the Constitutive Secretory Pathway" Journal of Biological Chemistry 266(19):12127-12130 (1991).
Hwang et al., "Why ION Pair Reversal by Protein Engineering is Unlikely to Succeed" Nature 334:270-272 (1988).
Knauf et al., "Relationship of Effective Molecular Size to Systemic Clearance in Rats of Reconbinant Interleukin-2 Chemically Modified with Water Soluble Polymers" The Journal of Biolgical Chemistry 29(Oct. 15):15064-15070 (1988).
Kraut, Joseph, "Serine Proteases: Structure and Mechanism of Catalysis" Ann. Rev. Biochem. 46:331-358 (1977).
Lipkind et al., "Molecular Modeling of the Substrate Specificity of Prohormone Convertases SPC2 and SPC3" The Journal of Biological Chemistry 270(22):13277-13284 (1995).
Matthews et al., "A Survey of Furin Substrate Specificity Using Substrate Phage Display" Protein Science 3:1197-1205 (1994).
Matthews et al., "X-ray Crystallographic Study of Boronic Acid Adducts with Subtilisin BPN' (Novo)" Journal of Biological Chemistry 250(18):7120-7126 (1975).
McPhalen et al., "Structural Comparison of Two Serine Proteinase-Protein Inhibitor Complexes: Eglin-C-Subtilisin Carlsberg and CI-2-Subtilisin Novo" Biochemistry 27:6582-6598 (1988).
Philipp et al., "Kinetics of Subtilisin and Thiolsubtilisin" Molecular and Cellular Biochemistry 51(5):5-32 (1983).
Poulos et al., "Polypeptide Halomethyl Ketones Bind to Serine Proteases as Analogs of the Tetrahedral Intermediate" The Journal of Biological Chemistry pp. 1097-1103 (1975).
Rheinnecker et al., "Engineering a Novel Specificity in Subtilisin BPN'" Biochemistry 32(5):1199-1203 (1993).
Rheinnecker et al., "Variants of Subtilisin BPN' with Altered Specificity Profiles" Biochemistry 33:221-225 (1994).
Robertus et al., "Subtilisin; a Stereochemical Mechanism Involving Transition-State Stabilization" Biochemistry 11:4293-4303 (1972).
Robertus et al., "An X-Ray Crystallographic Study of the Binder of Peptide Choloromethyl Ketone Inhibitors to Subtilisin BPN'" Biochemistry 11(13):2439-2449 (1972).
Russell et al., "Rational modification of enzyme catalysis by engineering surface charge" Nature 328:496-500 (1987).
Siezen et al., "Homology Modelling and Protein Engineering Strategy of Subtilases, the Family of Subtilisin-Like Serine Proteinases" Protein Engineering 4(7):719-737 (1991).
Siezen et al., "Homology Modelling of the Catalytic Domain of Human Furin, a Model for the Eukaryotic Subtilisin-Like Proprotein Convertases" FEBS Letter pp. 255-266 (1994).
Smeekens, Steven P., "Processing of Protein Precursors by a Novel Family of Subtilisin-Related Mammalian Endoproteases" Bio/Technology 11:182-186 (1993).
Stauffer et al., "The Effect on Subtilisin Actvity of Oxidizing a Methionine Residue" The Journal of Biological Chemistry 244(19):5333-5338 (1969).
Steiner et al., "The New Enzymology of Precursor Processing Endoproteases" Journal of Biological Chemistry 267(33):23435-23438 (1992).
Stennicke et al., "Effects of introduced aspartic and glutamic acid residues on the P'.sub.1 substrate specificity, pH dependence and stability of carboxypeptidase Y" Protein Engineering 7(7):911-916 (1994).
Svendsen, I., "Chemical Modifications of The Subtilisins With Specific Reference to the Binding of Large Substrates. A Review." Carlsberg Rs. Commun. 41(5):237-291 (1976).
Wells et al., "Cloning, sequencing, and secretion of Bacillus amyloliquefaciens subtilisin in Bacillus subtilis" Nucleic Acids Research 11(22):7911-7929 (1983).
Wells et al., "Designing substrate specificity by protein engineering of electrostatic interactions" Proc. Natl. Acad. Sci. USA 84:1219-1223 (1987).
Wise et al., "Expression of a human proprotein processing enzyme: correct cleavage of the von Willebrand factor precursor at a paired basic amino acid site" Proc. Natl. Acad. Sci. USA 87:9378-9382 (1990).
Wright et al., "Structure of Subtilisin BPN' at 2-5 A Resolution" Nature 221:235-242 (1969).
Estell et al., "Probing Steric and Hydrophobic Effects on Enzyme-Substrate Interactions by Protein Engineering" Science 233:659-663 (1986).
Graycar et al., "Altering the Proteolytic Activity of Subtilisin through Protein Engineering" Annals of the New York Academy of Sciences 672:71-79 (1992).
Nakayama et al., "Consensus Sequence for Precursor Processing at Mono-arginyl Sites" Journal of Biological Chemistry 267:16335-16340 (1992).
Wells et al., "Recruitment of Substrate-specificity Properties from One Enzyme Into a Related One by Protein Engineering" Proc. Natl. Acad. Sci. USA 84:5167-5171 (1987).
Wells et al., "Subtilisin: An Enzyme Made for Engineering" J. Cell. Biochem. (abstract B7-012) Suppl. 19B:233 (1995).
Ballinger Marcus D.
Wells James A.
Genentech Inc.
Kubinec Jeffrey S.
Moore William W.
Wax Robert A.
LandOfFree
Subtilisin variants capable of cleaving substrates containing di does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Subtilisin variants capable of cleaving substrates containing di, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Subtilisin variants capable of cleaving substrates containing di will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2056764