Ezrin-pIRES2-EGFP was constructed by subcloning full-length ezrin (1-586) into the Xho I and EcoR I sites of pIRES2-EGFP vector (Clonetech). The threonine 567 residue in ezrin was mutated to aspartic acid (T567D) using the QuikChange® II Site-Directed mutagenesis kit (Stratagene). In order to generate YFP fusions of Ezrin, the stop codon TAA in Ezrin-pIRES2-EGFP was mutated to GGA (Glycine), and the stop codon-mutated wild type ezrin was subcloned into the Xho I and EcoR I sites of the vector pEYFP-N1 (Clonetech). The T567 site in the resulting fusion construct (Ez-YFP) was mutated to aspartic acid to generate a T567D-YFP fusion protein (TD-YFP). 2PK3 cells were transfected with 3-6 μg of the appropriate plasmids using Amaxa Nucleofector II (Lonza). Stable transfectants were generated by selecting for G418-resistance and sorting for high GFP expression using the Aria I cell sorter (Becton Dickinson).
Protocol detail
Generating Ezrin Fusion Proteins
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Aria
Aspartic acid
Cell
Ezrin
G418
Glycine
Plasmids
Protein
Site directed mutagenesis
Stop codon
Taa codon
Threonine
Vector
Corresponding Organization :
Other organizations : Cleveland Clinic Lerner College of Medicine
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Variable analysis
independent variables
- Ezrin construct (Ezrin-pIRES2-EGFP, T567D-YFP, etc.)
- Protein expression (GFP/YFP)
- G418 resistance
- Transfection conditions (plasmid amount, cell line, transfection method)
- Cell sorting for high GFP expression
- Positive control: Wild-type Ezrin-pIRES2-EGFP
- Negative control: Not explicitly mentioned
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