title: Chemical synthesis and expression of a gene coding for human muscle acylphosphatase.
abstract: A DNA sequence coding for human muscle acylphosphatase has been constructed using 16 chemically synthesized oligonucleotides. The 300-bases long DNA sequence has been cloned in the pT7.7 Escherichia coli expression vector and in the pYEpsec1 Saccharomyces cerevisiae expression vector. In both cases a high level of expression of acylphosphatase has been observed. The recombinant proteins have been purified to homogeneity and assayed in comparison with the natural protein, using benzoylphosphate as a substrate and phosphate as a competitive inhibitor. The recombinant enzymes expressed in the two microorganisms maintain the kinetic properties of the natural protein. In addition, NMR analysis shows that the gross fold of the two recombinant enzymes is correct.
authors.affiliation: Department of Biochemical Science, University of Florence, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Modesti
authors.forename: G
authors.initials: G
authors.lastname: Raugei
authors.forename: N
authors.initials: N
authors.lastname: Taddei
authors.forename: R
authors.initials: R
authors.lastname: Marzocchini
authors.forename: M
authors.initials: M
authors.lastname: Vecchi
authors.forename: G
authors.initials: G
authors.lastname: Camici
authors.forename: G
authors.initials: G
authors.lastname: Manao
authors.forename: G
authors.initials: G
authors.lastname: Ramponi
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004247:DNA / Q000138:chemical synthesis* / Q000378:metabolism; D004926:Escherichia coli; D005813:Genes, Synthetic; D015186:Hominidae / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D000097024:Acylphosphatase
chemical_list: D017766:Acid Anhydride Hydrolases; D004247:DNA; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D000097024:Acylphosphatase