Expression, purification and crystallization of human 3'-phosphoadenosine-5'-phosphosulfate synthetase 1.
3'-Phosphoadenosine-5'-phosphosulfate (PAPS) is used to incorporate sulfate into biomolecules. The human PAPS synthetase 1 catalyzes two steps leading from adenosine triphosphate (ATP) and sulfate to PAPS. The ATP sulfurylase domain catalyzes the formation of the intermediate adenosine-5'-phosphosulfate (APS). The APS kinase domain then adds a phosphate group to the 3'-ribose and releases PAPS. In this article, the recombinant expression, purification and crystallization of the full-length protein is described. In Escherichia coli the protein is only partly soluble and copurifies with GroEL. The pure protein migrates as a dimer in gel-filtration chromatography. It is moderately active, forming 25 nmol PAPS per minute per milligram. Crystals grow to 100 x 100 x 300 micro m and diffract to 1.75 A.
Harjes
Stefan
S
Molekulare und Strukturelle Biophysik, Max-Planck-Institut für Molekulare Physiologie, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany. stefan.harjes@mpi-dortmund.mpg.de
Scheidig
Axel
A
Bayer
Peter
P
D000255:Adenosine Triphosphate / Q000737:chemistry; D018834:Chaperonin 60 / Q000737:chemistry; D002845:Chromatography; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006801:Humans; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D016622:Silver Staining; D013430:Sulfate Adenylyltransferase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D018834:Chaperonin 60; D018076:DNA, Complementary; D009097:Multienzyme Complexes; D006639:Histidine; D000255:Adenosine Triphosphate; C022322:PAPS synthetase; D013430:Sulfate Adenylyltransferase