Modified chitin-binding domain and use thereof
View Patent ↗Compositions and methods are provided for reversibly binding chitin binding domain (CBD) to a chitin or equivalent substrate under non denaturing conditions. CBD is modified preferably by a mutation to achieve this change in properties. In one embodiment, an aromatic amino acid residue located in the binding cleft of the CBD was altered resulting in reversible binding affinity for substrate in select conditions. Creating a modified CBD with an altered binding affinity for substrate provides new uses for CBD not previously possible with unmodified CBD which binds irreversibly to chitin.
1. A method for producing and purifying a target protein molecule comprising:
(a) constructing a DNA expression vector which expresses a hybrid polypeptide in a transformed host cell, the hybrid polypeptide comprising the target protein molecule and a modified chitin binding domain (CBD) that has a specific and reversible affinity for a substrate under non-denaturing conditions, wherein the non-denaturing conditions are selected from the group consisting of non-denaturing ionic conditions, non-denaturing pH, and non-denaturing detergent concentration;
(b) introducing the expression vector into an appropriate host cell and expressing the hybrid polypeptide;
(c) contacting the hybrid polypeptide produced by the transformed cell with the substrate to which the CBD binds; and
(d) recovering the hybrid polypeptide.
2. The method of claim 1 , wherein recovery of the hybrid polypeptide further comprises:
contacting the bound hybrid polypeptide with a substance which displaces the hybrid polypeptide.
3. A method according to claim 1 , wherein the altered non-denaturing ionic condition is a reduction in NaCl molarity from a salt concentration of 2M NaCl to a salt concentration in the range of 0–1M NaCl.
4. A method according to claim 1 , wherein the substrate is chitin.
5. A method for purifying a chitin binding domain-target molecule conjugate from a mixture of molecules in a preparation comprising:
(a) adding to the preparation, a substrate to which chitin binding domain (CDB) binds with a specific and reversible affinity under non-denaturing conditions, wherein the non-denaturing conditions are selected from the group consisting of non-denaturing ionic conditions, non-denaturing pH, and non-denaturing detergent concentration, so as to permit binding and immobilizing of the chitin binding domain-target molecule conjugate to the substrate;
(b) removing the bound chitin binding domain-target molecule conjugate from the mixture into a buffer; and
(c) recovering the purified chitin binding domain-target molecule conjugate.
6. A method according to claim 5 , wherein the altered non-denaturing ionic conditions comprise a reduction in NaCl molarity from a salt concentration of 2M NaCl to a salt concentration in the range of 0–1M NaCl.
7. A method according to claim 5 , wherein the substrate is chitin.