Protein Engineering Protocols - Mycobacteriology research center

100 Koide and Koidetarget-binding site in most cases, although we have also shown that the AB loopcan be used for target binding (10). Our method is based on the Bio-RadMutagene kit (17). Using E. coli SS320 for electrotransformation, a library containingapprox 10 9 independent clones can be readily prepared.3.1.1. Preparation of a Uracil-Single-Stranded Phagemid(for Monovalent Phage Display and Yeast Two-Hybrid Screening)1. Transform CJ236 with pAS47, pAS38 (for monobody–gene III) or pYT45 (forB42–monobody), select colonies on LB-Ap plate.2. Inoculate 2 mL of 2xYT-Ap-chloramphenicol with a colony; shake at 37°Covernight.3. Inoculate prewarmed 30 mL 2xYT-Ap supplemented with 0.25 µg/mL uridine ina 250-mL baffled flask with 300 µL of the overnight CJ236 culture, and add10 10 /mL helper phage KO7 (Promega). Grow at 37°C for 2 h with vigorous shaking.Add Km at a final concentration of 70 µg/mL. Grow overnight at 37°C with vigorousshaking.4. Centrifuge at 12,000g (10 krpm in a SS-34 [Sorvall] or equivalent rotor) for 10 min.Transfer supernatant to a new tube and centrifuge again at 12,000g for 10 min.Transfer supernatant to a new tube. Add 4.5 mL PEG/NaCl solution, and mix well.Incubate at 4°C overnight.5. Centrifuge at 17,200g (12 krpm, SS-34 rotor) for 20 min at 4°C. Discard supernatantand place the tube upside down on a stack of paper towels for 1 min. Wipeoff residual liquid with paper towel.6. Suspend the phagemid pellet in 1 mL TBS and transfer to a microcentrifuge tube.Centrifuge for 2 min at the maximal speed in a microcentrifuge. Transfer supernatantto a new microcentrifuge tube. Add 150 µL of PEG/NaCl solution and mixwell. Incubate on ice for 30 min.7. Centrifuge for 10 min at the maximum speed at 4°C. Discard supernatant, centrifugebriefly, and remove all liquid with a pipetter. Suspend the pellet in 1 mL TBS.8. Check the titers of the phagemid with XL-1 Blue and CJ236. Mix 100 µL of freshE. coli (optical density [OD] at 600 nm [OD 600nm] = 0.5–1.0) and 10 µL of a serialdilution of the phagemid solution. Incubate at room temperature for 15 min andplate on LB-Ap. Incubate the plates at 37°C overnight. The titer with CJ236 shouldbe 10 12 to 10 14 /mL and should be more than 10 4 higher than that with XL-1 Blue.9. Prepare uracil-single-stranded DNA using the QIAGEN single-stranded DNApreparation kit.3.1.2. Preparation of a Uracil-Single-Stranded Phage(for Multivalent Phage Display Vector, JCFN)1. Transfect CJ236 with JCFN plasmid. After heat shock, mix with 30 mL prewarmed2xYT and 500 µL of fresh CJ236 (OD 600nmof 0.5–1.0), and then shake at37°C for 6 h.2. Perform steps 4 to 9 of Subheading 3.1.1. For titering, mix 3 mL of LB and 0.7%agar (melted and cooled to 45°C), 10 µL of a serial dilution of the phage solution,