Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 49, Issue 2, Pages 228-234Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2006.04.001
Keywords
Na+-coupled HCO3; cotransporte; SLC4A4; purification; solubility; GroEL; copurification; secondary-structure; estimated molecular mass
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Funding
- NIDDK NIH HHS [R37 DK30344, F32 DK075258, R37 DK030344, K01 DK082646, 5T32 DK007259, R37 DK030344-28] Funding Source: Medline
- NINDS NIH HHS [R01 NS18400] Funding Source: Medline
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The cytoplasmic. N-terminal domain (Nt) of the electrogenic sodium/bicarbonate cotransporter-NBCel-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. (c) 2006 Elsevier Inc. All rights reserved.
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