Home › Biochemistry › Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae
Biochemistry JoVE (Open Access) Citable · DOI

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

DOI: 10.3791/51447-v
What you'll learn
  • ✓ Solubilize and purify CFTR protein from yeast microsomal membranes
  • ✓ Apply nickel-affinity and gel permeation chromatography purification methods
  • ✓ Reconstitute purified CFTR into lipid bilayers for functional assays
  • ✓ Measure CFTR ATPase activity to validate protein function
Protocol

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.

Difficulty
advanced
Total time
~3–5 days (including expression, lysis, multi-step chromatography, and functional assays)
Model organism
Saccharomyces cerevisiae
Biosafety
BSL-1

Steps

1
Solubilize microsomes containing CFTR protein

Extract yeast microsomes and solubilize membrane-bound CFTR using detergent-based lysis buffer. Clarify lysate by centrifugation to remove insoluble material.

▶ 01:46
2
Perform nickel-affinity purification of CFTR

Load clarified solubilized lysate onto immobilized-metal affinity chromatography (IMAC) column with nickel resin. Elute His-tagged CFTR using imidazole gradient.

▶ 03:49
3
Polish CFTR using gel permeation chromatography

Apply IMAC-purified CFTR to size-exclusion chromatography column to remove aggregates and further isolate monodisperse protein. Collect peak fractions.

▶ 05:33
4
Reconstitute purified CFTR into lipid bilayers

Dialyze purified CFTR against lipid-containing buffer to facilitate insertion into proteoliposomes. Remove detergent by gradual dialysis.

▶ 06:57
5
Measure CFTR ATPase activity in vitro

Assess reconstituted CFTR function using ATPase assays. Quantify ATP hydrolysis rates to confirm protein activity.

▶ 07:51
6
Validate milligram-scale CFTR isolation by two methods

Compare yield and purity of CFTR obtained via nickel-affinity and gel permeation chromatography routes using SDS-PAGE and mass spectrometry.

▶ 09:35
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