Abstract
AFFINITY chromatography has been used extensively for purification of enzymes by specific, reversible binding of the active site to a substrate analogue coupled to an insoluble matrix1. Escherichia coli β-galactosidase has been purified by this method2. The enzyme is composed of four identical subunits of molecular weight 135,000, each containing an active site3. Many Z (β-galactosidase) gene mutant strains are available which produce only a portion of the polypeptide chain. These polypeptides, which are enzymatically inactive, cross-react with antibody prepared to the wild type enzyme4. We have used affinity chromatography to determine whether incomplete polypeptide chains from a variety of Z gene mutants are folded in the conformation necessary for recognition of substrate. Specific alkylation with a substrate analogue was also used as a test for the presence of a substrate binding site. The use of mutant chains containing different regions of the β-galactosidase sequence made it possible to determine which portions of the molecule are most directly involved in the formation of the binding site.
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VILLAREJO, M., ZABIN, I. Affinity Chromatography of β-Galactosidase Fragments. Nature New Biology 242, 50–52 (1973). https://doi.org/10.1038/newbio242050a0
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DOI: https://doi.org/10.1038/newbio242050a0
