AFFINITY CHROMATOGRAPHIC PURIFICATION OF ANTIBODIES TO A BIOTINYLATEDFUSION PROTEIN EXPRESSED IN ESCHERICHIA-COLI

HOP1, a protein component of the synaptonemal complex in Saccharomyces cerevisiae which is believed to play an important role in meiotic syn apsis, was expressed in Escherichia coli as a fusion protein incorpora ting a ''tag'' polypeptide which is biotinylated naturally in the bact eria. The HOP1 fusion protein was produced in an insoluble form within the bacteria; once solubilized using a denaturing agent, the protein was purified by avidin monomer affinity chromatography. The recombinan t protein was used to immunize rabbits and produce polyclonal antibodi es, Procedures for affinity purification of antibodies using the recom binant protein attached to the avidin column and a magnetic method for concentration of antibodies are described. Antibody elution condition s in these procedures do not affect the affinity of the column for the recombinant protein, which can be recovered afterward, Affinity-purif ied antibodies show high binding capacity to HOP1 recombinant protein in immunoblotting experiments, but reduced background compared with cr ude antiserum or purified IgG fraction. The affinity-purified antibodi es recognize a major band around 70 kDa in Western blots of yeast prot ein extracts following meiotic induction. (C) 1998 Academic Press.