Back to results
US 5,856,201 AGranted
Methods of detection using a cellulose binding domain fusion product
AssigneesYissum Research Development Company of the Hebrew University of Jerusalem, University of California
InventorsOded Shoseyov, Itai Shpiegl, Marc A. Goldstein, Roy H. Doi
Life of this patent
Estimating the term…
Abstract
A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.
Claims29
A method of detecting the presence of a substance of interest in a test sample comprising:
- (a) incubating a test sample, which may contain a substance of interest, with a sufficient amount of a cellulose binding domain (CBD) fusion product comprising (i) a cellulose binding domain, the amino acid sequence of which has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:2, and which is capable of binding to cellulose or chitin with high affinity and is substantially free of other proteins with which it is naturally associated, and (ii) a second protein capable of binding said substance of interest, under conditions that allow for the binding of said substance of interest to said second protein of said cellulose binding domain fusion product;
- (b) contacting the test sample and cellulose binding domain fusion product with an amount of cellulose or chitin effective to bind the amount of said cellulose binding domain fusion product used in step (a) to provide an insoluble cellulose-cellulose or chitin-cellulose binding domain fusion product binding complex;
- (c) separating said insoluble cellulose-cellulose or chitin-cellulose binding domain fusion product binding complex from unbound components;
- (d) incubating said insoluble cellulose-cellulose or chitin-cellulose binding domain fusion product binding complex with a sufficient amount of a detectable label, said label capable of binding to said substance of interest;
- and
- (e) separating said insoluble cellulose-cellulose or chitin-cellulose binding domain fusion product binding complex of step (d) from unbound components and determining the presence or absence of said label, to provide an indication of the presence or absence of said substance of interest in said test sample.
3 more elements
A method of detecting the presence of a substance of interest in a test sample comprising:
- (a) contacting a test sample, which may contain a substance of interest, with an insoluble matrix capable of immobilizing said substance of interest;
- (b) incubating said insoluble matrix with a sufficient amount of a cellulose binding domain fusion product comprising (i) a cellulose binding domain (CBD), the amino acid sequence of which has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:2, and which is capable of binding to cellulose or chitin with high affinity and is substantially free of other proteins with which it is naturally associated, and (ii) a second protein capable of binding said immobilized substance of interest, under conditions that allow for the binding of said immobilized substance of interest to said second protein of said cellulose binding domain fusion product;
- (c) separating said insoluble matrix of step (b) from unbound components;
- (d) incubating said insoluble matrix of step (c) with a detectable label capable of binding said substance of interest or said cellulose binding domain fusion product or cellulose or chitin under conditions that allow for the binding of said label to said substance of interest or said cellulose binding domain fusion product or cellulose or chitin;
- and
- (e) separating said insoluble matrix of step (d) from unbound components and determining the presence or absence of said label, to provide an indication of the presence or absence of said substance of interest in said test sample.
3 more elements
A method of detecting the presence of a substance of interest in a test sample comprising:
- (a) incubating a test sample, which may contain a substance of interest, with a sufficient amount of a cellulose binding domain (CBD) fusion product comprising (i) a cellulose binding domain, the amino acid sequence of which has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:2, and which is capable of binding to cellulose or chitin with high affinity and is substantially free of other proteins with which it is naturally associated, and (ii) a second protein capable of binding said substance of interest, under conditions that allow for the binding of said substance of interest to said second protein of said cellulose binding domain fusion product;
- (b) contacting the test sample and cellulose binding domain fusion product with an amount of cellulose or chitin effective to bind the amount of said cellulose binding domain fusion product used in step (a) to provide an insoluble cellulose-cellulose or chitin-cellulose binding domain fusion product binding complex;
- (c) separating said insoluble cellulose-cellulose or chitin-cellulose binding domain fusion product binding complex from unbound components;
- (d) incubating said insoluble cellulose-cellulose or chitin-cellulose binding domain fusion product binding complex with a sufficient amount of a detectable label comprising a labeled substance of interest, said label capable of binding to any second protein of said cellulose binding domain fusion product which remains unbound to said substance of interest;
- and
- (e) separating said insoluble cellulose-cellulose or chitin-cellulose binding domain fusion product binding complex of step (d) from unbound components and comparing the signal observed from said test sample relative to the signal observed from a control sample.
3 more elements
Dependencies are inferred from the claim text.