Protein Engineering Protocols - Mycobacteriology research center

Engineered M13 Bacteriophage Coat Proteins 217target. After washing to remove unbound phage, bound phage are detected spectrophotometrically(at 450 nm), using a reaction catalyzed by a horseradish peroxidase/M13antibody conjugate. An increase in hGH display is indicated by anincrease in the efficiency of hGH-P8 phage capture. From a plot of the phageconcentration vs absorbance at 450 nm, the increase in hGH display with a P8variant relative to that with wt P8 can be estimated by comparing the phage concentrationsrequired to generate a particular absorbance at 450 nm (11). Clonesexhibiting high hGH display are subjected to DNA sequence analysis to determinethe sequences of the P8 variants within the hGH-P8 fusions.1. From a fresh LB/tet plate, pick a single colony of E. coli XL-1 Blue harboring theappropriate phagemid into 1 mL of 2YT medium supplemented with M13KO7helper phage (10 10 pfu/mL), 50 µg/mL carbenicillin (for phagemid maintenance),and 5 µg/mL tetracycline (for F′ episome maintenance). Shake at 200 rpm and37°C for 2 h and add 25 µg/mL kanamycin to select for clones that have beencoinfected with M13KO7. Shake at 200 rpm and 37°C for 6 h and transfer the cultureto 30 mL of 2YT/carb/kan medium. Shake overnight at 200 rpm and 37°C.2. Centrifuge for 10 min at 27,000g and 4°C (15 krpm in a Sorvall SS-34 rotor).Transfer the supernatant to a new tube containing 1/5 volume of PEG/NaCl andincubate for 5 min at room temperature. Centrifuge 10 min at 12,000g and 4°C(10 krpm in an SS-34 rotor). Decant the supernatant. Centrifuge briefly at 2000g(4 krpm) and aspirate the remaining supernatant.3. Resuspend the phage pellet in 0.5 mL of PBS-T-BSA buffer and transfer to a1.5-mL microcentrifuge tube. Centrifuge for 5 min at 14,000g in a microcentrifuge,and transfer the supernatant to a 1.5-mL microcentrifuge tube.4. Determine the phage concentration spectrophotometrically (ε 268= 1.2 × 10 8 /M/cm).5. Prepare fivefold serial dilutions of phage stock, using PBS-T-BSA buffer.6. Transfer 100 µL of phage solution to Maxisorp immunoplates coated with hGHbpand blocked with BSA (see Subheading 3.3.1.1.). Incubate for 1 h with gentleshaking.7. Remove the phage solution and wash eight times with PBS-T buffer.8. Add 100 µL of horseradish peroxidase/M13 antibody conjugate (diluted 3000-foldin PBS-T-BSA buffer). Incubate for 30 min with gentle shaking.9. Wash eight times with PBS-T buffer and two times with PBS.10. Develop the wells with 100 µL of TMB substrate. Stop the reaction with 100 µLof 1.0 M H 3PO 4, and read spectrophotometrically at 450 nm in a microtiter platereader.4. Notes1. For mutagenesis to introduce the stop codons, the protocols in Subheadings3.2.2.2 and 3.2.2.3 can be scaled down 10-fold. The reaction product can be useddirectly to transform E. coli using any standard procedure.2. The protocol can be scaled up by inoculating a larger overnight culture and purifyingthe ssDNA with multiple spin columns.