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
Volume37
Issue2
Pagination479-85
Date Published2004 Oct
ISSN1046-5928
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
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.

DOI10.1016/j.pep.2004.06.035
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