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Protein Expr Purif. 2006 Oct;49(2):228-34. Epub 2006 Apr 25.

Expression and purification of the cytoplasmic N-terminal domain of the Na/HCO3 cotransporter NBCe1-A: structural insights from a generalized approach.

Author information

1
Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA. hs.gill@yale.edu

Abstract

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.

PMID:
16757179
DOI:
10.1016/j.pep.2006.04.001
[Indexed for MEDLINE]

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