Overexpression of the Saccharomyces cerevisiae MET17/MET25 gene in Escherichia coli and comparative characterization of the product with O-acetylserine.O-acetylhomoserine sulfhydrylase of the yeast.
The Saccharomyces cerevisiae MET17/MET25 gene encoding O-acetyl-L-serine (OAS).O-acetyl-L-homoserine (OAH) sulfhydrylase (EC 4.2.99.10) was overexpressed in Escherichia coli and the gene product was purified to homogeneity, using three steps, with a recovery of 28% from the total cell extract. The gene product has been compared with OAS.OAH sulfhydrylase purified from the yeast cells. These two protein preparations were indistinguishable with respect to their behavior in polyacrylamide gel electrophoresis, both with and without sodium dodecyl sulfate, their specificity for substrate amino acids, Michaelis constant (Km) value for OAH, sensitivity to carbonyl reagents, absorption spectrum, isoelectric point, behavior in HPLC (both ion-exchange chromatography and gel filtration), sensitivity to heat treatment, susceptibility to trypsin digestion, and their N-terminal amino acid sequence. The results obtained imply that the gene product is properly processed in E. coli, and the technique developed in this study to overexpress the gene in bacterial cells provides us with a large amount of the purified preparation of the enzyme. In contrast to a previous report we found that cystathionine gamma-lyase of S.
Yamagata
S
S
Department of Biology, Faculty of General Education, Gifu University, Japan.
Isaji
M
M
Nakamura
K
K
Fujisaki
S
S
Doi
K
K
Bawden
S
S
D'Andrea
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D001709:Biotechnology; D019757:Carbon-Oxygen Lyases; D003001:Cloning, Molecular; D003547:Cysteine Synthase; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005800:Genes, Fungal; D008190:Lyases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity
D017931:DNA Primers; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D003547:Cysteine Synthase; C103381:MET17 protein, S cerevisiae; C018725:O-acetylhomoserine (thiol)-lyase; D008190:Lyases; D019757:Carbon-Oxygen Lyases