Ni-chelate-affinity purification and crystallization of the yeast mitochondrial F1-ATPase.

TitleNi-chelate-affinity purification and crystallization of the yeast mitochondrial F1-ATPase.
Publication TypeJournal Article
Year of Publication2004
AuthorsMueller, DM, Puri, N, Kabaleeswaran, V, Terry, C, Leslie, AGW, Walker, JE
JournalProtein Expr Purif
Date Published2004 Oct
KeywordsAdenosine Triphosphatases, Cell Proliferation, Chloroform, Chromatography, Affinity, Chromatography, Gel, Codon, Crystallography, X-Ray, Electrophoresis, Polyacrylamide Gel, Fungal Proteins, Histidine, Mitochondria, Models, Genetic, Protein Structure, Secondary, Protein Structure, Tertiary, Proton-Translocating ATPases, Saccharomyces cerevisiae

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.

Alternate JournalProtein Expr. Purif.
Citation Key10.1016/j.pep.2004.06.035
PubMed ID15358374
Grant List1F06TW002379 / TW / FIC NIH HHS / United States
R01-GM066233 / GM / NIGMS NIH HHS / United States
R01-GM067091 / GM / NIGMS NIH HHS / United States