Expression and purification of the cytoplasmic N-terminal domain of the Na/HCO3 cotransporter NBCe1-A: structural insights from a generalized approach.
The cytoplasmic, N-terminal domain (Nt) of the electrogenic sodium/bicarbonate cotransporter--NBCe1--over-expresses in Escherichia coli and yields a large amount of soluble protein. A novel purification strategy, which involves a streptomycin precipitation, overcomes obstacles of instability and copurifying proteins, and leads to the first seen Nt-NBCe1 crystals. The purification procedure generally lends itself to the purification of Nts from other classes of the SLC4 family. Size-exclusion chromatography suggests that the Nt of NBCe1 as well as the Nt of other SLC4 members form dimers. A comparison of Nt-NBCe1 to SLC4 member Nt-AE1, based on purification properties and predicted secondary-structure sequence alignments, suggests a similar mechanism for dimer stabilization.
Gill
Harindarpal S
HS
Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA. hs.gill@yale.edu
Boron
Walter F
WF
D019281:Dimerization; D006801:Humans; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D027982:Sodium-Bicarbonate Symporters / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
D011994:Recombinant Proteins; D027982:Sodium-Bicarbonate Symporters