[Expression and purification of ATP sulfurylase from Saccharomyces cerevisias in Escherichia coli and its application in pyrosequencing].
ATP sulfurylase (ATPS,EC 2.7.7.4) reversibly catalyzes the reaction between ATP and sulfate to produce APS and pyrophosphate (PPi), and has been used in pyrosequencing. The gene coding ATP sulfurylase was amplified from the genomic DNA of Saccharomyces cerevisias (CICC 1202), and cloned into prokaryotic expression plasmid pET28a( + ) to provide a recombinant expression plasmid pET28a( + )-ATPS. Upon IPTG induction, ATP sulfurylase was produced by E. coli BL21 (DE3) harboring the recombinant expression plasmid pET28a( + )-ATPS. The relative molecular weight of recombinant ATP sulfurylase with His tag was about 60 kD. The recombinant ATP sulfurylase with electrophoretic pure grade was obtained only by two purification steps: His * Bind Resin affinity chromatography and ultrafiltration. The specific activity of the purified recombinant ATP sulfurylase was as high as 5.1 x 10(4) u/mg. The successful application of the enzyme in pyrosequencing was also demostrated.
Luo
Juan
J
College of Life Science and Technology, China Pharmnaceutical University, Nanjing 210009, China.
Wu
Wen-Juan
WJ
Zou
Bing-Jie
BJ
Zhou
Guo-Hua
GH
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*; D013430:Sulfate Adenylyltransferase / Q000096:biosynthesis* / Q000235:genetics