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

  • Gill H
  • Boron W
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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. © 2006 Elsevier Inc. All rights reserved.

Author-supplied keywords

  • Estimated molecular mass
  • GroEL copurification
  • Na+-coupled HCO3-cotransporter
  • Purification
  • SLC4A4
  • Secondary-structure
  • Solubility

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Authors

  • Harindarpal S. Gill

  • Walter F. Boron

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