Xie, B., Tassi, E., Swift, M.R., McDonnell, K., Bowden, E.T., Wang, S., Ueda, Y., Tomita, Y., Riegel, A.T. and Wellstein, A.
Notes: Starting with the fibroblast growth factor-binding protein (FGF-BP1) open reading frame cloned into the SP72 Vector, 5’ and 3’ deletions were generated in the gene to use in phage library display. For the 5’ deletion library, BamHI was used to generate the 5’ overhang, which is susceptible to exonuclease III treatment, while KpnI generated a 3’ overhang to protect the plasmid backbone. To create the 3’ deletion library, NotI and ApaI restriction sites were introduced into pSP72-FGF-BP1 Vector with NotI close to the 3’ end of FGF-BP1. NotI was used to generate the 5' overhang and ApaI generated a 3’ overhang. Exonuclease III was added at 20°C with an estimated erasing rate at around 90bp/minute. Enzymatic digestion of plasmids was stopped at 30-second intervals. Digested plasmids were then pooled, religated and cloned at the EcoRI and HindIII sites into the phage 10-3b vector. In vitro packaging generated the stock library solution, which was then used to localize the interaction interface between FGF-2 and FGF-BP1. (3508)