Protein Engineering Protocols - Mycobacteriology research center

216 Sidhu et al.3.3. Selection and Analysis of P8 Variants That IncreaseFusion Protein Display3.3.1. Selection of Phage From the hGH-P8 LibraryPhage from the hGH-P8 library described above are cycled through roundsof binding selection with hGHbp (16) coated on 96-well Maxisorp immunoplatesas the capture target. Phage are propagated in E. coli XL-1 Blue withM13KO7 helper phage for further rounds of selection.3.3.1.1. COAT WELLS WITH TARGET PROTEIN1. Coat 8 wells of a 96-well Maxisorp plate with 100 µL of a 5 µg/mL solution ofthe target protein (e.g., hGHbp) overnight at 4°C. Discard the solution by emptyingthe plate contents into the sink.2. To block nonspecific binding to the microtiter wells, add 200 µL of 0.2% BSA inPBS to each well. Shake at room temperature for 1 h.3.3.1.2. PHAGE LIBRARY SELECTION1. Add a solution of the phage library (~10 12 phage/mL) in PBS-T-BSA buffer to thewells. Shake at room temperature for 2 h. Wash the plate eight times with PBS-Tbuffer. The stringency of the binding selection can be increased for successiverounds by increasing the number of washes.2. Elute bound phage, by adding 100 µL of 100 mM HCl to each well. Shake vigorouslyfor 5 min at room temperature.3. Combine the eluants and neutralize with 1/5 volume of 1.0 M Tris-base.3.3.1.3. PROPAGATE PHAGE FOR FURTHER ROUNDS OF SELECTION1. Add the eluted phage to 10 volumes of XL-1 Blue cells (OD 550= 0.5–1.0).2. Shake at 200 rpm and 37°C for 20 min and remove 10 µL for titers, as describedin Subheading 3.2.3., step 4.3. Add M13KO7 helper phage and shake at 200 rpm and 37°C for 45 min.4. Transfer the culture to 100 mL 2YT/carb/kan medium and shake overnight at200 rpm and 37°C.5. Isolate the phage by PEG/NaCl precipitation, as described in Subheading 3.2.3,steps 8 through 10.6. Repeat the selection process five times, using only half of the eluted phage in eachround.3.3.2. Phage Enzyme-Linked Immunosorbent Assay Screen to AssessLevels of hGH DisplayAfter selection for hGH display (Subheading 3.3.1.), individual clones areanalyzed in a phage enzyme-linked immunosorbent assay (2,11) to assess levelsof hGH display relative to display with wt P8. Serial dilutions of hGH-P8 phagesolutions are incubated in wells containing immobilized hGHbp as the capture