Bicyclic peptide phage library production

  1. Inoculate 1L 2YT media with the phage library cell stock to reach OD600 of 0.1.
  2. Produce the phage library at 30°C with 230rpm shaking for 16hours.
  3. Centrifuge the culture at 6000rpm for 30mins at 4°C, and collect the supernatant.
  4. At ? volume of PEG/NaCl solution (20% PEG-6000 (w/v), 2.5 M NaCl), and mix well.
  5. Incubate the upper mixture solution in ice for another one hour by shaking the bottle every another 15 mins.
  6. Pellet the phage by centrifugation at 7000rpm for 45 mins. Dump the supernatant and collect the phage pellet.
  7. Suspend the phage pellet in 10mL buffer R (20mM NH4HCO3, 5mM EDTA)
  8. Add 1mM TCEP to the upper phage solution and reduce the phage at 42°C for 1 hour.
  9. Remove excess TCEP by concentrating the phage solution in vivaspin colume (100K cutoff) to 1mL.
  10. Add 3mL well degassed buffer R and spin the solution to 1mL. repeat this step for another two times.
  11. Add 8mL buffer R the remaining 1mL phage solution and transfer the phage solution to a new falcon tube.
  12. Add 2mL of 100uM TBMB in ACN to the phage solution to perform the bicyclic modification.
  13. Incubate the modification reaction mixture at 30°C for 1 hour.
  14. Add 2.5mL PEG/NaCl solution to precipitate the modified phage.
  15. Resuspend the bicyclic peptide phage library in a suitable buffer for the target protein supplemented with 1% BSA, 0.1% Tween-20.