title: Cloning, expression and purification of arylsulfate sulfotransferase from Eubacterium A-44.
abstract: A gene (astA) encoding arylsulfate sulfotransferase (ASST), which transfers a sulfate group from phenolic sulfate esters to phenolic acceptors, was cloned from a Eubacterium A-44 genomic library. The probe (1.5 kb fragment) for the astA gene was prepared from the PCR product of the primers produced using two internal amino acid sequences of ASST, which had been purified from Eubacterium A-44. The astA gene was cloned into the pKF3 vector. Its sequence revealed a 1863 bp open reading frame (ORF) encoding a protein containing 620 amino acids with a secretary signal peptide, and showed 91% homology (identity) to Eubacterium rectale IIIH previously reported. The cloned astA gene was expressed under the T7 promoter of the expression vectors, pET-39b(+) and pET-26b(+), in Escherichia coli BL21 (DE3), and the expressed ASSTs were purified using His Bind column chromatography. The specific activities of the purified ASSTs were 25.6 micromol/min/mg and 37.1 micromol/min/mg, respectively.
authors.affiliation: College of Pharmacy, Kyung Hee University, Seoul, Korea.
authors.forename: Bomi
authors.initials: B
authors.lastname: Kim
authors.forename: Yang-Jin
authors.initials: YJ
authors.lastname: Hyun
authors.forename: Keun-Sook
authors.initials: KS
authors.lastname: Lee
authors.forename: Kyoichi
authors.initials: K
authors.lastname: Kobashi
authors.forename: Dong-Hyun
authors.initials: DH
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D015239:Arylsulfotransferase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005051:Eubacterium / Q000201:enzymology*; D015723:Gene Library; D007218:Industrial Microbiology / Q000379:methods; D008969:Molecular Sequence Data; D010226:Parabens / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D017423:Sequence Analysis, RNA; D013379:Substrate Specificity; D014169:Transformation, Bacterial; D014439:Tyramine / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D010226:Parabens; D011994:Recombinant Proteins; D014443:Tyrosine; D015239:Arylsulfotransferase; C090707:arylsulfate sulfotransferase; C038193:4-hydroxybenzoic acid; D014439:Tyramine