Cloning, expression and purification of arylsulfate sulfotransferase from Eubacterium A-44.
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.
Kim
Bomi
B
College of Pharmacy, Kyung Hee University, Seoul, Korea.
Hyun
Yang-Jin
YJ
Lee
Keun-Sook
KS
Kobashi
Kyoichi
K
Kim
Dong-Hyun
DH
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
D001426:Bacterial Proteins; D010226:Parabens; D011994:Recombinant Proteins; D014443:Tyrosine; D015239:Arylsulfotransferase; C090707:arylsulfate sulfotransferase; C038193:4-hydroxybenzoic acid; D014439:Tyramine