title: Ni-chelate-affinity purification and crystallization of the yeast mitochondrial F1-ATPase.
abstract: The yeast mitochondrial ATPase has been genetically modified to include a His(6) Ni-affinity tag on the amino end of the mature beta-subunit. The modified beta-subunit is imported into the mitochondrion, properly processed to the mature form, and assembled into a mature and fully active ATP synthase. The F(1)-ATPase has been purified from submitochondrial particles after release from the membrane with chloroform, followed by Ni-chelate-affinity and gel filtration chromatography. The final enzyme is a homogeneous preparation with full activity and no apparent degradation products. This enzyme preparation has been used to obtain crystals that diffract to better than 2.8 A resolution.
authors.affiliation: Department of Biochemistry and Molecular Biology, The Chicago Medical School, USA. David.Mueller@rosalindfranklin.edu
authors.forename: David M
authors.initials: DM
authors.lastname: Mueller
authors.forename: Neeti
authors.initials: N
authors.lastname: Puri
authors.forename: Venkataraman
authors.initials: V
authors.lastname: Kabaleeswaran
authors.forename: Cassandra
authors.initials: C
authors.lastname: Terry
authors.forename: Andrew G W
authors.initials: AG
authors.lastname: Leslie
authors.forename: John E
authors.initials: JE
authors.lastname: Walker
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry; D049109:Cell Proliferation; D002725:Chloroform; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003062:Codon; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D008928:Mitochondria / Q000201:enzymology*; D008957:Models, Genetic; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D006180:Proton-Translocating ATPases / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism
chemical_list: D003062:Codon; D005656:Fungal Proteins; D006639:Histidine; D002725:Chloroform; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases