Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Stablizing an enzyme by forming a mixture – an adduct or a...
Reexamination Certificate
1999-04-22
2001-02-06
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Stablizing an enzyme by forming a mixture, an adduct or a...
C435S183000
Reexamination Certificate
active
06184012
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to constructs and methods for the generation, detection, and isolation of enzymes possessing a desired chemical or biological activity. In particular it relates to a method for the generation, detection and isolation of enzymes or proteins with enzyme-like activity, with a novel substrate or reaction specificity or with improved activity. In addition, the invention permits the subsequent identification and isolation of the nucleic acid encoding the enzyme.
BACKGROUND OF THE INVENTION
A number of different approaches have hitherto been taken to generate novel polypeptides with new, modified, or improved biological activity. For example, the alteration of individual residues at the active site of enzymes of known crystallographic structure (Winter et al., 1982; Wilkinson et al., 1984), immunization with transition state analogues (Tramontano et al., 1986) the screening of mutant microorganisms harbouring new properties (Cunningham and Wells, 1987), the alteration of specific residues within a signal peptide window and the generation of novel antibodies by phage display (Marks et al., 1994) have all led to the generation of polypeptides with tailored functions.
Antibody fragments have been expressed bacterially and in bacteriophage (phage) display repertoires by fusion to phage coat proteins, in particular the minor coat protein cpIII. Subsequent selection of phage with antigen has allowed the isolation of high affinity antibodies from large libraries without immunisation.
The endogenous genetic machinery of phage, and the ability to generate a large population of individually unique phage clones means that a huge (>10
8
) variety of different recombinant polypeptides can be produced. A large number of variants of a certain polypeptide chain are cloned into the genome of filamentous phage or constructed as phagemids and expressed as a fusion protein with the phage coat protein. This library is then selected by panning to the ligand of interest; after extensive washings and elution, those phages expressing a binding variant are rescued by infection of bacteria. Multiple rounds of selection allow the isolation of very rare phage (<1/10
7
).
Phage display has also been used as a tool to investigate the relative efficacy of signal peptides, to evaluate the individual contribution of each residue of an epitope, and to refine the properties of such biological molecules. For example, phage display has been used to isolate zinc finger domains with altered DNA-binding specificity, improved hormones and novel inhibitors.
The generation of novel enzymes, or enzymes with improved function has proven more difficult. The main obstacles in this case are associated with methods of selection. In nature, new enzymes arise through random mutation and Darwinian selection. Initial attempts to mimic this process used mutant microorganisms, selecting for increased enzyme activity by growth advantage (Cunningham and Wells, 1987). More recently, both alkaline phosphatase and trypsin have been displayed on the surface of phage, and have been shown to retain their catalytic activity (McCaf ferty et al., 1991, Corey et al., 1993). Such phages have been enriched by binding to suicide inhibitors that bind irreversibly to the protein. Soumillion et al., 1994 describe incubation of phage displaying a &bgr;-lactamase with a &bgr;-lactamase suicide inhibitor connected by a spacer to a biotin moiety. Active phages were selected by binding and elution from streptavidin-coated beads. Such a procedure suffers from the drawback that a suicide inhibitor or a transition state analogue must be available for the reaction of interest. This is not generally the case. Moreover, indirect selections result in low rate accelerations. A direct selection for the desired catalytic activity would yield better results.
SUMMARY OF THE INVENTION
According to the present invention, there is provided a method of isolating an enzyme having a desired chemical or biological activity the method comprising:
a) linking the enzyme to a substrate for the enzyme;
b) reacting the enzyme with the substrate such that a product is produced which remains linked to the enzyme; and
c) isolating the active enzyme by selectively isolating the product linked to the enzyme.
The invention also provides a method of isolating DNA encoding an enzyme comprising a) linking the enzyme to a substrate for the enzyme; b) reacting the enzyme and substrate such that a product is produced which remains linked to the enzyme; and c) isolating the enzyme by selectively isolating the product linked to the enzyme; wherein the enzyme is further linked to DNA encoding the enzyme.
According to a further aspect of the present invention there is also provided a composition comprising an enzyme linked to a substrate for the enzyme or linked to a product of the reaction between the enzyme and the substrate.
REFERENCES:
patent: 5807718 (1998-09-01), Joyce et al.
patent: 5910408 (1999-06-01), Szostak et al.
patent: 7-126297 (1995-05-01), None
patent: WO 95/12672 (1995-05-01), None
patent: WO 96/17086 (1996-06-01), None
patent: WO 96/40723 (1996-12-01), None
patent: WO 97/09446 (1997-03-01), None
P. Soumillion et al., 1994, Selection of Beta-Lactamase on Filamentous Bacteriophage by Catalytic Activity, J. Mol. Biol., 237:415-422.
M. Illangasekare, et al., Feb. 1995, Aminoacyl-RNA Synthesis Catalyzed by an RNA, Science, 267:643-647.
J.R. Lorsch and J.W. Szostak, 1994, In Vitro Evolution of New Ribozymes with Polynucleotide Kinase Activity, Nature, 371:31-36.
D.S. Tawfik, et al, 1993, Catelisa: A Facile General Route to Catalytic Antibodies, Proceedings of the Nat'l. Acad. of Sci., 90:373-377.
McCafferty, et al., 1991, Phage-enzymes: Expression and Affinity Chromatography of Functional Alkaline Phosphatase on the Surface of Bacteriophage, Protein Engineering, 4:995-961.
Corey, et al., 1993, Trypsin Display on the Surface of Bacteriophage, Gene 128:129-134.
Demartis Salvatore
Huber Adrian
Neri Dario
Tawfik Dan S
Viti Francesca
Darby & Darby
Medical Research Council
Weber Jon P.
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