Protein Protocols Protein Protocols

Phage Display 1065
3.1.3.2. AFFINITY SELECTION
1. Incubate phage display library (1–2 × 10 11 pfu) with 0.1 µg of biotinylated target (see
Subheading 3.1.1.2.) in 500 mL of biopan buffer overnight at 4°C.
2. Add phage display library–target mixture to Streptavidin coated plates.
3. Incubate at room temperature for 10 min.
4. Add 0.2 mM biotin in biopan buffer (500 µL) to displace any Streptavidin-binding phage.
5. Incubate an additional 5 min.
6. Proceed from step 4 in Subheading 3.1.2.2. (see Note 8).
3.2. Phage Display on Proteins Expressed in Whole Cells
3.2.1. Target Generation
3.2.1.1. INFECTION FOR BIOPANNING ON INSECT CELLS
1. Seed 1–2 × 106 cells in 1 mL of EX-CELL 401 medium containing 1% Fetal bovine
serum into 35-mm plates (one well/infection).
2. Let cells adhere for 1 h at 27°C.
3. Add 0.5–5 MOI Baculovirus (for target and control) to medium.
4. Grow for 48–72 h at 27°C.
5. Harvest Baculovirus-infected cells into 15-mL polypropylene tube(s).
3.2.1.2. TRANSFECTION FOR BIOPANNING ON MAMMALIAN CELLS
Transient transfections can be performed using Lipofectamine to make a complex
with the target cDNA and incubating the complex with the desired cell line. Satisfactory
results can be obtained using target cDNA in pCDNA3 (Invitrogen) (29).
1. Seed 1–3 × 105 cells/well in 2 mL of complete DMEM, in a six-well tissue culture plate.
2. Grow cells at 37°C in an atmosphere of 5% CO2–95% air to 80% confluence.
3. Use 3 µg of DNA and 8 mL of Lipofectamine for each transfection.
4. Dilute DNA and Lipofectamine separately in serum- and antibiotic-free DMEM
(100 µL/transfection).
5. Combine solutions with gentle mixing.
6. Incubate at room temperature for 30 min.
7. Meanwhile, wash cells with 2 mL of serum- and antibiotic-free DMEM.
8. Add 0.8 mL of serum- and antibiotic-free DMEM, for each transfection, to the DNA–lipid
mixture.
9. Mix gently.
10. Carefully place DNA–lipid mixture over rinsed cells (1 mL total volume).
11. Incubate complex mixture with cells at 37°C in an atmosphere of 5% CO2–95% air for 5 h.
12. Add 1 mL of DMEM, 20% fetal bovine serum to each transfection mixture after 5 h incubation.
13. Incubate complex mixture at 37°C in an atmosphere of 5% CO2–95% air for 18 h.
14. Replace medium with 2 mL of complete DMEM.
15. Collect cells after an additional 24-h incubation at 37°C in an atmosphere of 5%
CO2–95% air.
3.2.2. Affinity Selection by Biopanning Against Control (Uninfected)
and Infected/Transfected Whole Cells
There are at least two published protocols that describe the use of phage display
targeting proteins expressed as cell surface receptors in whole cells (30,31). The following
method is an adaptation of that described by Goodson et al. (30), who discovered
novel urokinase receptor antagonists with phage display (see Note 9).