The Purification of Chloramphenicol Acetyltransferae from Saccharomyces Cerevisiae Harboring the Escheria Coli Chloramphenicol Resistance Gene
Thompson, Lorri J.
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Chloramphenicol acetyl transferase was purified to homogeneity from stationary phase Saccharomyces cerevisiae transformed by the plasmid pYT11. This plaslmd is a composite vector consisting of the bacterial plasmid pBR325 , the yeast 2µ plasmid and a restriction fragment carrying the yeast LEU2 structural gene. The relevant genotype of this plasmid is cam r. Purification was achieved by ammonium sulfate precipitation and affinity column chromatography. The affinity column material was prepared by attaching the ligand, chloramphenicol base, to Sepharose gel using methods described by Zaidenzaig and Shaw (1976), Er-el et al (1972) and March et al (1974). Purity of the chloramphenicol acetyl transferase was determined by the increase of enzymatic activity per mg of the purified enzyme over the crude enzyme extract.and by SDS polyacrylamide gel electrophoresis. A seven hundred fold increase in specific activity of chloramphenicol acetyl transferase was attained by affinity column chromatography as determined by a spectroyhotometric assay. The specific activity of the bacterially produced enzyme was previiously found to be 240 units/mg as compared to the specific activity of 296 units/mg of the eukaryotically produced enzyme as determined during this project . The purity and molecular weight of a subunit of chloramphenicol acetyltransferase were determined by SDS polyacrylamide gel electrophoresis. Purification was judged visually by a comparison of' the band produced by the crude enzyme extract in an SDS polyacrylamide gel to the band produced by the purified enzyme. The band produced by the purified enzyme was narrow and distinct whereas the band produced by the crude enzyme extract was wide and indistinct. The molecular weight of a subunit of chloramphenicol acetyl transferase was found to be 31,000 daltons as compared to the molecular weight of 20,000 daltons for the bacterially produced enzyme.