Kathleen Cornely

The HICA project part of CUREnet:CURE Collection
In this CURE, inspired by the work of Hoffmann, et al., students prepare mutant Haemophilus influenzae carbonic anhydrase (HICA) proteins. Using PyMOL to visualize the three-dimensional structure of the HICA protein, students choose one or more surface amino acid residues to mutate to histidine residues in order to create a surface histidine cluster that will allow the mutant protein to bind to a nickel affinity column. Using site-directed mutagenesis, recombinant plasmids are constructed and are then used to transform an E. coli expression vector. The mutant HICA protein is overexpressed, cells are lysed, and students load the cell lysate onto Ni-NTA columns and determine the imidazole concentration required to elute the mutant protein. The construction of a library of mutant proteins will allow the development of a general method in which specific surface histidine residues of any protein can be mutated in order to facilitate affinity purification. The Haemophilus influenzae bacterium described herein is a respiratory pathogen that causes meningitis (in its encapsulated form) and mucosal infections such as otitis media, sinusitis and conjunctivitis (in its unencapsulated form). A recent study showed that the carbonic anhydrase enzyme is absolutely required for pathogenesis. Furthermore, expression of the HICA enzyme allows the pathogen to survive in host immune cells (Langereis, et al.). These observations make the study of HICA itself particularly attractive, in addition to the overall goal of contributing to a body of work that will allow the minimal histidine character required for nickel affinity to be ascertained.