Subject Area
Chemistry
Description
Carbonic anhydrase (CA) is an enzyme that plays a major role in the survival of many bacterial, chiefly Haemophilus influenzae. Because of its crucial role in bacteria, recent research has turned to CA as a possible target for drug development to kill bacteria and possibly cure different bacterial diseases. While research has focused on this drug target, the isolation and purification of specific types of CA has remained a major obstacle for further research. The current method of immobilized metal affinity chromatography (IMAC) with a Ni-NTA column is used widely for CA purification; however, the H. influenzae carbonic anhydrase (HICA) has very low binding affinity to Ni-NTA column and cannot be effectively purified. Previous research has shown that the addition of a surface histidine residue at D125 increases HICA protein binding affinity to the nickel column.
In our research, we suggest that the further insertion of surface histidine residues will increase the HICA protein binding affinity to a Ni-NTA column. The addition of the Ile126His and Lys129His mutations to the Asp125His HICA mutant through site directed mutagenesis is presented here to observe the binding affinity of the mutant HICA to a nickel column, The newly synthesized HICA mutant is reported here to enhance the binding affinity of HICA to the Ni-NTA column, eluting from the column upon addition of between 50 and 100 mM imidazole, as opposed to wild-type HICA protein that elutes upon the addition of 25 mM imidazole. To confirm the presence of the mutant HICA protein in the 50 and 100 mM fractions, SDS-PAGE analysis was used and it is reported here that molecular weight of the monomer of the protein was 19.6 kDa. These promising results indicate that the addition of multiple histidine residues to the HICA enzyme can increase its binding affinity to a Ni-NTA column. As opposed to adding polyhistidine tags to the HICA protein, these results suggest a straightforward strategy to purify the HICA enzyme as well as other proteins for further research in drug development.
Publisher
Providence College
Date
2020
Type
Article
Format
Text
Language
English
