title: Cloning, overexpression, and purification of cytosine deaminase from Saccharomyces cerevisiae.
abstract: Cytosine deaminase is an enzyme which has been investigated for cancer chemotherapy as a result of its ability to convert the relatively nontoxic prodrug 5-fluorocytosine into the anticancer drug 5-fluorouracil. To facilitate investigations of the utility of cytosine deaminase for cancer chemotherapy, we have cloned and expressed the enzyme from Saccharomyces cerevisiae. The DNA sequence translates into a protein of 158 amino acids in length, with a predicted molecular weight of 17,563 kilodaltons. Alignment of the cytosine deaminase protein sequence from yeast with a variety of proteins defines a novel sequence motif of cytosine or cytidine binding enzymes. Recombinant expression cassettes encoding cytosine deaminase were transfected into monkey kidney COS cells, which lack endogenous cytosine deaminase, to test for production of a functional protein. Cell extracts from these transfectants contained detectable levels of enzyme activity capable of converting 5-fluorocytosine to 5-fluorouracil. Cytosine deaminase was expressed in yeast from a cDNA cassette under the control of an inducible promoter, increasing expression 250- to 300-fold relative to wild-type strains. A purification protocol has been developed which permits recovery of 60% of cytosine deaminase in active form from induced cell lysates after two purification steps. This protocol will be useful for isolating large quantities of pure enzyme which are required for the preclinical evaluation of monoclonal antibody-cytosine deaminase conjugates in combination with 5-fluorocytosine.
authors.affiliation: Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121, USA.
authors.forename: M S
authors.initials: MS
authors.lastname: Hayden
authors.forename: P S
authors.initials: PS
authors.lastname: Linsley
authors.forename: A R
authors.initials: AR
authors.lastname: Wallace
authors.forename: H
authors.initials: H
authors.lastname: Marquardt
authors.forename: D E
authors.initials: DE
authors.lastname: Kerr
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D003001:Cloning, Molecular / Q000379:methods*; D043525:Cytosine Deaminase; D004790:Enzyme Induction / Q000187:drug effects; D005690:Galactose / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D008715:Methionine / Q000032:analysis / Q000378:metabolism; D008969:Molecular Sequence Data; D009700:Nucleoside Deaminases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D016415:Sequence Alignment; D013462:Sulfur Radioisotopes; D013997:Time Factors
chemical_list: D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D008715:Methionine; D009700:Nucleoside Deaminases; D043525:Cytosine Deaminase; D005690:Galactose