Production Of A Mutant, Recombinant FAR Protein Library For Use In Future Structure-Function Studies Of The Nematode-Specific FAR Family Of Protein.

Abstract

We have mutagenized the hp-far-1 gene, which encodes for the Hp-FAR-1 protein, a fatty acid and retinol binding protein. This protein is secreted by the nematode parasite Heligmosomoides polygyrus, a model used in the study of chronic helminth infections. We have generated both maintenance and protein expression cell lines to express normal and mutant Hp-FAR-1 forms. Point mutations were introduced to change hydrophobic residues to serine. The normal and mutant proteins were expressed with 6x His tags using IPTG induction, and isolated using nickel affinity chromatography. Both the normal and mutant proteins were present in the soluble fractions and were present in column fractions at greater than or equal to 90% purity. Purity and molecular weight were analyzed by SDS- PAGE. The recombinant proteins were identified by western blot analysis using commercially purchased anti-His tag antibody. Contaminating detergents were removed using detergent removing Extracti-D gel resin column chromatography. The mutant proteins are currently undergoing spectrophotometric analysis to identify any reductions and/or loss of function in their ability to bind fatty acids and retinol, as compared to the non-mutated protein.