RESUMO
The yeast enzyme phenylacrylic acid decarboxylase (PAD) confers resistance to phenylacrylic acids. Cinnamic acid (CA)-sensitive mutants lacking PAD activity were isolated and the PAD1 gene was cloned by phenotypic complementation. The nucleotide sequence of the smallest complementing fragment was determined. The predicted 242-amino-acid PAD polypeptide is 48.6% identical to the product of dedF of Escherichia coli. PAD activity and CA resistance, but not steady-state PAD1 mRNA levels, are influenced by mitochondrial genotype. PAD1 is a single-copy gene in the yeast genome and not essential for viability. The PAD1 locus was physically mapped to a position approx. 140 kb from the left end of chromosome IV.
Assuntos
Carboxiliases/genética , Cinamatos/farmacologia , Proteínas Fúngicas/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Fúngico , Resistência Microbiana a Medicamentos/genética , Dados de Sequência Molecular , Mapeamento por Restrição , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologiaRESUMO
An economical method to express recombinant protein in yeast (Saccharomyces cerevisiae) was developed. We combined two principles: Ni(2+)-agarose-based affinity purification of fusion proteins and expression from a cassette regulated by steroid hormones. After induction by desoxycorticosterone, > 90% pure protein was obtained after a single round of metal affinity chromatography. Gentle lysis conditions allowed the preservation of enzymatic activity. Average yields of 10 mg protein per liter of culture were obtained at a fraction of the cost of other eukaryotic expression systems.