Modified chitin binding domain and uses 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 molecular conjugate, comprising: a recombinant chitin binding domain (CBD) linked to a molecule of interest, the chitin binding domain containing at least one mutation such that the mutated chitin binding domain is capable of elution from a chitin substrate under a selected non-denaturing condition in which the chitin binding domain without the at least one mutation remains bound.
2. A conjugate according to claim 1 , wherein the at least one mutated amino acid is an aromatic amino acid.
3. A conjugate according to claim 1 , wherein the at least one mutated amino acid is positioned within a binding cleft of the CBD.
4. A conjugate according to claim 2 , wherein the at least one mutated amino acid comprises a phenylalanine in place of a tryptophan.
5. A conjugate according to claim 4 , wherein the tryptophan corresponds to tryptophan 687 of Bacillus circulans chitinase A1, identified as tryptonhan 33 of SEQ ID NO: 1.
6. A conjugate according to claim 1 , wherein the selected non-denaturing condition is selected from the group consisting of non-denaturing ionic conditions, non-denaturing pH, and non-denaturing detergent concentration.
7. A conjugate according to claim 1 , wherein the selected non-denaturing condition comprises reducing the salt concentration from 2M NaCI to a salt concentration in the ranoe of 0-1M NaCI.
8. A conjugate according to claim 1 , wherein the chitin binding domain is a bacterial chitin binding domain.