Ni-chelate-affinity purification and crystallization of the yeast mitochondrial F1-ATPase.
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.
Mueller
David M
DM
Department of Biochemistry and Molecular Biology, The Chicago Medical School, USA. David.Mueller@rosalindfranklin.edu
Puri
Neeti
N
Kabaleeswaran
Venkataraman
V
Terry
Cassandra
C
Leslie
Andrew G W
AG
Walker
John E
JE
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
D003062:Codon; D005656:Fungal Proteins; D006639:Histidine; D002725:Chloroform; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases