Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1997-01-31
2002-12-31
Saidha, Tekchand (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S069100, C435S069700, C435S252300, C435S320100, C435S442000, C435S007100, C536S023200, C536S023700, C530S300000, C530S350000
Reexamination Certificate
active
06500660
ABSTRACT:
BACKGROUND OF THE INVENTION
The development of assays for measuring the presence and amount of desired substances is highly desirable for a variety of purposes, including for medical, veterinary, research, and environmental uses. It is further desirable to design and isolate molecules having an activity which is regulatable by a desired substance. Assays can then be designed to detect the amount and presence of a desired substance, such as an analyte in a test sample, utilizing the ability of the analyte to directly or indirectly (e.g., by competition) regulate the molecule's activity. Assays can then be designed which utilize these regulatable activities.
DESCRIPTION OF THE INVENTION
The present invention relates to a chimeric target molecule having an activity which can be regulated or modulated by a binding molecule. The invention also relates to methods of using the chimeric target molecule to detect the presence and/or amount of a desired analyte in a sample. The analyte is a binding molecule, or a competitor of a binding molecule, which binding molecule, upon binding to the target molecule, alters the activity of the target molecule in a detectable way. In one aspect of the invention, a binding molecule binds to the chimeric molecule, inactivating it. An analyte in a test sample competes and/or displaces the binding molecule from the chimera, reactivating it. The reappearance of activity in the presence of the analyte indicates its existence and amount in the test sample. Another aspect of the invention relates to a binding molecule which regulates a chimeric target molecule and methods of producing it.
In accordance with the present invention, a desired target molecule (TM) can be modified to have at least one binding site moiety (BSM) to which a binding molecule (BM) can attach. Upon attachment of the BM to the BSM, an activity associated with the TM is altered in a detectable way, e.g., increasing or reducing the activity of the TM. Thus, the BSM can act as a regulatory switch, turning on or off (all or in part) an activity of a desired TM in response to the binding of a BM. The BSM can also be selected so that binding of the binding molecule regulates the activation of the target molecule. In accordance with the present invention, a mimetope is the preferred BSM. A BSM can be engineered into a target molecule by the insertion of sequences, by the replacement of sequences present in the molecule with new sequences, by mutagenesis of sequences already present in the molecule, etc. Engineering can be accomplished according to methods available to the skilled worker.
The target molecule can be selected for a desired detectable activity. For example, the TM can be: &bgr;-lactamase: P. Soumillion et al.,
J. Mol. Biol
., 237:415:-422, 1994; Plasmin: L. Jespers et al., conference communication; Prostate specific antigen: R. Ecrola et al.,
Biochem. Biophys. Res. Comm
., 200:1346-1352, 1994; Subtilisin: P. Soumillion et al.,
Appl. Biochem. Biotechnol
., 47:175-190, 1994; Trypsin: D. R. Corey et al.,
Gene
, 128:129-134, 1993; Alkaline phosphatase: J. McCafferty et al.,
Prot. Enging
., 4:955-961; &bgr;-galactosidase: I. N. Maruyama et al.,
Proc. Natl. Acad. Sci. USA
, 91:8273-8277, 1994; Staphylococcal nuclease: J. Ku & P. G. Schultz, Bioorg.
Med. Chem
., 2:1413-5, 1994; and J. Light & R. A. Lerner,
Bioorg. Med. Chem
., 3:955-67, 1995; Glutathione transferase: M. Widersten & B. Mannervick,
J. Mol. Biol
., 250:115-122, 1995; Lysozyme: K. Maenaka et al.,
Biochem. Biophys. Res. Comm
., 218:682-687, 1996; and Catalytic antibodies: K. D. Janda et al.,
Proc. Natl. Acad. Sci USA
, 91:2532-2536, 1994.
The above-mentioned target molecules have been displayed on phage. They are directly amenable to the method of selection of BSM. Other enzymes can also be displayed on phage and are useful for the present invention, e.g., esterases, pyruvate kinase, glucose oxidase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, luciferase. The TM can also be a protein possessing a fluorescent activity (e.g., green fluorescent protein, GFP: Chalfie et al., 1994
, Science
, 263:802; Cheng et al., 1996
, Nature Biotechnology
, 14:606; Levy et al., 1996
, Nature Biotechnology
, 14:610) which is modulated by binding of a BM to a BSM contained within the fluorescent protein. The TM can also be a regulatory molecule which activates/inactivates a second molecule having a detectable activity. For instance, a GTPase activating protein (GAP) stimulates a G-protein, such as ras. The ability of a GAP to activate a G-protein can be modulated by engineering a BSM into the GAP. Upon attachment of a BM to the BSM of a modified GAP, the stimulating activity of the GAP can be modulated. Its upstream effect on G-proteins can be monitored, e.g., by measuring a GTPase activity of the G-protein. See, e.g., Trahey and McCormick,
Science
, 238:542-545, 1987. The TM can also be a subunit of another protein which itself possesses enzymatic or another detectable activity. Additionally, the TM can be a nucleic acid enzyme, e.g., a ribozyme, a hammerhead enzyme, RNAse P, or a hairpin enzyme. If a nucleic acid is used as the target molecule, the engineered binding site moiety would usually comprise nucleotides, either modified or naturally-occurring. The TM can also be transcription activators and repressors regulated in in vitro transcription and translation systems; detection of activity can be accomplished at the level of the activity of the expressed enzyme or fluorescent molecule.
The activation of a chimeric molecule can also be regulated by a BM. The simplest example of activation is the proteolytic cleavage of a peptide bond in a zymogen to transform it into an enzyme. A classical example is the activation of a serine protease, or more specifically the activation of chymotrypsinogen into chymotrypsin by proteolytic cleavage of the peptide bond Arg15-Ile16 by trypsin. An antibody binding to an epitope or a mimotope engineered in the region of the cleaved peptide bond can inhibit the activation. Another example is the inhibition of the phosphorylation or dephosporylation of an enzyme whose activity is regulated by its state of phosphorylation. Glycogen phosphorylase is an example: when it is phosphorylated on Ser14, it is essentially in its active form, dephosphorylation deactivates the enzyme. Binding of an antibody to a engineered epitope or mimotope in the vicinity of the phosphorylation site would interfere with the activation/deactivation mechanism by phosphorylase kinase and phosphoprotein phosphatase respectively.
More generally any postraductional modification of an enzyme, that contributes to modulate its activity, can be interfered with by binding a foreign molecule to a BSM (e.g., an antibody).
The term “chimeric” target molecule, e.g., a “chimeric enzyme,” means the resultant product after the binding site moiety has been inserted into the target molecule or after a portion of the target molecule has been replaced by the binding site moiety. For clarity, before engineering of the BSM, the target molecule is referred to as the starting target molecule. Thus, if an enzyme is the starting material, it is referred to as the “starting enzyme.” After engineering of the BSM, the starting enzyme is identified as a “chimeric enzyme.” In the examples below, &bgr;-lactamase is used as a starting enzyme into which a binding site moiety comprising amino acids, is engineered to produce a chimeric enzyme. It is chimeric because it is comprised of amino acids of the starting enzyme and amino acids of a binding site moiety.
Target and chimeric molecules can be prepared by methods which are available in the art. For example, genetic engineering can be employed to prepare target and chimeric molecules which comprise amino acid or nucleotide residues. In one embodiment, a cloned gene is employed as the starting material for the starting target molecule and resultant chimeric target molecule. In the examples described below, the cloned gene for the starting enzyme &bgr;-lactamase serves as the beginning materi
Evans, ESQ Barry
Kramer Levin Naftalis & Frankel LLP
Saidha Tekchand
Universite Catholique de Louvain
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