Libraries phage

Incubate cells at 37 C for 30 min without shaking and then for 30 min with moderate shaking (200 r.p jn.) to allow infection. 

Transfer cultures to 250 ml centrifuge pots and centrifuge to pellet the cells (e.g. Sorvall RC-5B rotor, 5000 r.p.m., 10 min). Decant off the supernatant, resuspend the bacterial pellet in 250 ml of 2TY (no glucose) with 100 pg/ml ampicillin and 50 p,g/ ml kanamycin, and transfer to ftesh flasks. Grow overni t with rapid shaking (300 r.p.m.)at30°C. 

Since the phage titre after overnight growth is usually aroimd 1 x 10 /ml it is generalfy sufficient to precipitate phage f rom only 100 ml of the overui t culture. Pellet the cells from 100 ml culture (50 ml from each flask) (e.g. Sorvall RC-5B rotor. 8000 r.p.m., 15 min). 

Decant as much supernatant as possible, brie re-sjm the tubes, ai remove residual supernatant with a pipette. Resuspoid each phage pellet in 1 ml of IE and pool the ph e to give a 10 ml phage stock. 

Centrifuge the phage to remove any remaining bacterial debris (e.g. Sorrall SM 24 rotor, 8000 r.pjn., 5 mia) and transfer the supernatant to a fresh tube. This k your purified and concentrated phage stock which can be used in a first round of selection. It is stable for approx, one week if kept at 4 °C The phage titre should be approx. 1 X 10 /ml. 

---

Barry MA, Dower WJ, Johnston SA. Toward cell-targeting gene therapy vectors selection of cell-binding peptides from random peptide-presenting phage libraries. Nat Med 1996 2(3) 299-305. 

FIGURE 5.7 Schematic representation of the construction of a phage library. 

In conclusion, the ProxiMol technique may enable us to form the maps of multiprotein complexes as it provides both targeting specific cell surface receptors and other proteins associated with this receptor. No matter what technique is used to form phage libraries, it is not possible to interpret the data manually it is accomplished by using bioinformatics. 

The phage library stock may now be stored at 4°C. However, the selection procedure must be carried out with freshly prepared phage, since the antibody fusion protein may not be stable. To do this, the library can be reamplified by growth on XLl-Blue (Section 3.2.7., steps 7—17). 

Remove the blocking solution, add 100 jjL of freshly prepared phage library stock/well, and incubate at 37°C for 2 h. 

Positional scanning with phage libraries were used to discover the amino acid residues in peptides responsible for binding to the calciumbinding protein calmodulin.18 In this case, the binding polypeptide needed Trp as a key residue located in the fourteenth position from the N-terminus for strong binding. 

Garrard, L. J. and Henner, D. J. (1993) Selection of an anti-IGF-1 Fab from a Fab phage library created by mutagenesis of multiple CDR loops. Gene 128, 103-109. 

---