Partial purification of the cystic fibrosis transmembrane conductance regulator

L. S OSTEDGAARD; M. J WELSH

doi:10.1016/S0021-9258(18)35728-4

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Partial purification of the cystic fibrosis transmembrane conductance regulator

Journal article

Open access

Peer reviewed

Partial purification of the cystic fibrosis transmembrane conductance regulator

L. S OSTEDGAARD and M. J WELSH

The Journal of biological chemistry, Vol.267(36), pp.26142-26149

1992

DOI: 10.1016/S0021-9258(18)35728-4

PMID: 1281484

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Abstract

We have investigated several purification strategies for the cystic fibrosis transmembrane regulator (CFTR) based on its structural similarity to other proteins of the traffic ATPase/ABC transporter family. Recombinant CFTR expressed in heterologous cells was readily solubilized by digitonin and initially separated from the majority of other cellular proteins by sucrose density gradient centrifugation. CFTR, with two predicted nucleotide binding domains, bound avidly to several triazine dye columns, although elution with MgATP, MgCl2, or high ionic strength buffers was inefficient. CFTR did not bind to either ATP or ADP coupled to agarose. Because CFTR is a glycoprotein we investigated its binding to lectin columns. CFTR bound readily to wheat germ agglutinin, but poorly to Lens culinaris agglutinin. CFTR was enriched 9-10 times when eluted from wheat germ agglutinin with N-acetylglucosamine. This enrichment was tripled if lectin chromatography followed sucrose gradient centrifugation. Our results suggest the combination of sucrose density gradient centrifugation and lectin chromatography would be a satisfactory approach to initial purification of CFTR expressed in heterologous cells.

Fundamental and applied biological sciences. Psychology

Proteins

Binding and carrier proteins

Biological and medical sciences

Analytical, structural and metabolic biochemistry

Details

Title: Subtitle: Partial purification of the cystic fibrosis transmembrane conductance regulator
Creators: L. S OSTEDGAARD - Univ. Iowa coll. medicine, Howard Hughes medical inst., dep. internal medicine, Iowa City IA 52242, United States
M. J WELSH - Univ. Iowa coll. medicine, Howard Hughes medical inst., dep. internal medicine, Iowa City IA 52242, United States
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.267(36), pp.26142-26149
DOI: 10.1016/S0021-9258(18)35728-4
PMID: 1281484
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: American Society for Biochemistry and Molecular Biology; Bethesda, MD
Language: English
Date published: 1992
Academic Unit: Neurology; Molecular Physiology and Biophysics; Neurosurgery; Internal Medicine
Record Identifier: 9984020868102771

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