Protein Engineering Protocols - Mycobacteriology research center

Monobodies 105a reporter gene nor interact with wild-type FNfn10. This can be easily testedusing the interaction mating method (13,19).1. Transform RFY206 with a LexA-bait plasmid (or pEG202 for a negative control,and pBait [Origene] for a positive control) and pSH18-34 (β-galactosidase reporterplasmid; Origene), and select on YC Glc his– ura– plates. Also transform EGY48with pYesTrp2 (activator B42 plasmid; Invitrogen), pTarget (positive control;Origene), or pYT45, and select on YC Glc trp– plates.2. Streak two colonies each with a LexA plasmid and the reporter plasmid on a YCGlc his– ura– plate as parallel lines. Streak colonies with B42 plasmid on a YCGlc trp– plate in the same manner. Incubate both plates at 30°C for 1 to 2 d.3. Replicate the plates to a single YPD plate. The lines from the two plates should beperpendicular to each other so that EGY48 cells and RFY206 cells mate at eachoverlap. Incubate at 30°C overnight.4. Replicate the YPD plate to YC Glc leu– his– trp– ura–, YC Glc his– trp– ura–, andYC Gal Raf leu– his– trp– ura– plates, and incubate at 30°C for 3 d. All matedcells should grow on YC Glc his– trp– ura– plates. Growth on the YC Gal Raf leu–his– trp– ura– plate indicates interaction between the bait and prey. The otherplates are for controls.3.3.2. Yeast Two-Hybrid Library ScreeningRFY206 containing a bait is mated to EGY48 containing a monobodylibrary, and cells with a monobody that bind to the bait are selected. The use ofyeast mating makes it possible to efficiently screen multiple libraries (13).1. Grow RFY206 harboring pSH18-34 and a LexA-bait plasmid in 10 mL of YC Glchis– ura– media at 30°C for 16 h.2. Determine the cell density at OD 600nmusing 1.0 OD 600nm= 2.0 × 10 7 cells/mL.Spin down 10 8 bait cells in a microcentrifuge tube for 30 s at 10 krpm in a microcentrifuge,and suspend the yeast cells in 200 µL of the media.3. Add 10 7 EGY48 cells harboring a monobody to the bait cells (Subheading 3.1.7.),and plate them onto a YPD plate. Allow the cells to mate for 16 h.4. Wash the cells off the plate with 5 mL (1 mL at a time) of YPD media. Measurethe OD 600nmof the cells after a 100-fold dilution, and plate 10 8 cells on five YCGal Raf leu– his– trp– ura– plates. Incubate the plates for 3 to 4 d at 30°C.5. Replicate colonies onto YC Gal Raf leu– his– trp– ura– plates and YC Glc leu–his– trp– ura– plates. Incubate the plates for 16–24 h. True positive clones shouldgrow only on YC Gal Raf leu– his– trp– ura– plates.6. In a chemical hood, open the lid of the plate, and add enough chloroform to coverall of the surface of the plate. After 5 min of exposure, decant the chloroform intoa waste container and let the remaining chloroform on the plate evaporate for10 min (20).7. Heat the agarose–phosphate solution (10 mL per plate) in a microwave to dissolvethe agarose, and cool to 50°C in a water bath. In a test tube, add 35 µL of 50 mg/mLX-gal solution and 10 mL agarose–phosphate solution, vortex, and pour onto the