Protein Engineering Protocols - Mycobacteriology research center

104 Koide and Koideup to 1 mM for higher induction of monobody) and incubate with vigorous shakingat 37°C for 6 h (longer incubation may lead to a deletion within the monobodygene). Determine the titer immediately after adding 2xYT, as described inSubheading 3.1.2. For phagemid, mix 5 mL of fresh XL-1 Blue and elutedphagemid, and incubate for 20 min at 37°C. Add 100 mL of 2xYT (and IPTG, ifdesired), 100 µg/mL Ap, and 10 10 /mL helper phage KO7; incubate with vigorousshaking at 37°C for 2 h. Add Km at a final concentration of 70 µg/mL and incubateovernight. Determine the titer immediately after the 20 min incubation, as describedin Subheading 3.1.1. Isolate the phage (or phagemid) as described in Subheadings3.1.1. and 3.1.2. Repeat the entire procedure until there is an increase in the elutedphage(mid) number.3.2.2. Characterization of Individual Phage Clones1. Prepare the phage(mid) from a single clone. For the phagemid, grow a singlecolony in 2 mL of LB-Ap overnight. Mix 20 µL of the culture, 2 mL of LB-Ap(and IPTG, if desired), and 10 10 /mL helper phage KO7. Incubate overnight at37°C with vigorous shaking. For phage clones, touch a single plaque with a steriletoothpick. Place the toothpick in 2 mL of LB-Ap (and IPTG, if desired) with 100 µLof fresh XL-1 Blue, and incubate at 37°C with vigorous shaking for 6 h. Prepare thephage(mid) as described in Subheadings 3.1.1. and 3.1.2.2. For each clone to be tested, prepare a well with an immobilized target by performingsteps 1 and 2 from Subheading 3.2.1. Also prepare a control well by coating withcoating solution without the target (see Note 6).3. To the ligand-coated and the control wells, add 5 µL of 5% BSA solution and 50 µLof phage suspension prepared from individual clones. Adjust the phage concentration(typically 10 8 –10 11 /well) depending on the strength of interaction. Incubate atroom temperature for 2 h on a rotator.4. Wash five times with TBST. Using a squirt bottle, add TBST to all of the wells,then shake off the liquid and slap the plate face down against a stack of papertowels.5. To each well, add 50 µL of antibody to phage–horseradish peroxidase solution(Pharmacia; diluted 1:2000 in TBST:BSA), incubate 1 h at room temperature ona rotator. Wash five times with TBST.6. Add 50 µL of 1step turbo-TMB (Pierce), wait approx 10 min until blue colordevelops, and add 50 µL of 2 M H 2SO 4to stop the reaction (use filtered tips to protectthe pipetter). Measure at OD 450nmusing a plate reader. Select clones that showhigh binding to the ligand-coated well and low binding to the control well for furthercharacterization (sequencing, affinity maturation, and protein production).3.3. Yeast Two-Hybrid Screening3.3.1. Preparation of a Target (“Bait”) PlasmidClone the gene of a bait in pEG202 so that the bait is expressed in frame withLexA. It is important to make sure that the LexA-bait does not self-activate