Abstract
We have developed procedures to purify highly functional recombinant cystic fibrosis transmembrane conductance regulator (CFTR) from Chinese hamster ovary (CHO) cells to high homogeneity. Purification of CHO-CFTR was achieved using a combination of alkali stripping, alpha-lysophosphatidylcholine extraction, DEAE ion-exchange, and immunoaffinity chromatography. Insect CFTR from Sf9 cells was purified using a modification of the method of Bear et al. (Bear, C. E., Li, C., Kartner, N., Bridges, R. J., Jensen, T. J., Ramjeesingh, M. and Riordan, J. R. (1992) Cell 68, 809-818), which included extraction with sodium dodecyl sulfate, hydroxyapatite, and gel filtration chromatography. Characterization of the properties of purified CFTR from both cell sources using a variety of electrophysiological and biochemical assays indicated that they were very similar. Both the purified CHO-CFTR and Sf9-CFTR when reconstituted into planar lipid bilayers exhibited a low pS, chloride-selective ion channel activity that was protein kinase A- and ATP-dependent. Both the purified CHO-CFTR and Sf9-CFTR were able to interact specifically with the nucleotide photoanalogue 8-N3-[alpha-32P]ATP with half-maximal binding at 25 and 50 microM, respectively. These values compare well with those reported for 8-N3-[alpha-32P]ATP binding to CFTR in its native membrane form. Thus CFTR from either insect or CHO cells can be purified to high homogeneity with retention of many of the biochemical and electrophysiological characteristics of the protein associated in its native plasma membrane form. The availability of these reagents will facilitate further investigation and study of the structure and function of CFTR and its interactions with cellular proteins.
Highlights
From the Genzyme Corporation, Framingham, Massachusetts 01701-9322 and the §Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1XB Canada
We have developed procedures to purify highly functional recombinant cystic fibrosis transmembrane conductance regulator (CFTR) from Chinese hamster ovary (CHO) cells to high homogeneity
CFTR from either insect or CHO cells can be purified to high homogeneity with retention of many of the biochemical and electrophysiological characteristics of the protein associated in its native plasma membrane form
Summary
Vol 270, No 28, Issue of July 14, pp. 17033-17043, 1995 Printed in U.S.A. Purification and Characterization of Recombinant Cystic Fibrosis Transmembrane Conductance Regulator from Chinese Hamster Ovary and Insect Cells*. CFTR from either insect or CHO cells can be purified to high homogeneity with retention of many of the biochemical and electrophysiological characteristics of the protein associated in its native plasma membrane form. The availability of these reagents will facilitate further investigation and study of the structure and function of CFTR and its interactions with cellular proteins. The most common CF-associated mutation, a deletion of the residue phenylalanine at position 508 (~F508) prevents both the transport and maturation of the glycoprotein to its apical location (Cheng et al, 1990; Kartner et al, 1992; Denning et al, 1992a, 1992b) In consequence, these epithelial cells are unable to regulate normal ion transport, and this, it is proposed, is the basis for the disease. The feasibility of this approach has been extended to include the use of purified and reconstituted CFTR (generated using the processes described here) in either a proteoliposome or hybrid virosome.f
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