834525.fig.001
Figure 1: Schematic representation of the inverse SF-link selection method for screening of CatE-activity-enhancing peptides at neutral pH. A DNA template construct for primary peptide library with SF-link is shown where YLBS-generated variable region is integrated with T7 promoter, POU-domain, His-tag, Factor Xa sequences at upstream part, and further elongated with CatE substrate, FLAG, and PLBR (Puromycin linker binding region) sequences at downstream part (step 1). DNA template library is transcribed into mRNA library followed by ligation with Puromycin-linker DNA at the 3′-terminus end (step 2) and then converted into cDNA-displayed SF-link peptide products which is composed of both the functional part (random peptide sequence) and CatE peptide substrate (step 3). Finally, inverse SF-link selection (step 4) was performed using cDNA-tagged SF-link peptide library: (a) the nonspecific binding peptides were removed by NHS-activated sepharose beads. (b) The CatE binding peptides were recovered at 25°C after 10 min. of incubation with CatE-bound beads. (c) CatE-binding peptides were subjected to protease (CatE) cleavage at 37°C with cutoff incubation time in three successive rounds. (d) The rapidly cleaved peptides (still attached to cDNA) were recovered, PCR-treated, and used for the next selection round. (e) Molecules selected were subjected to cloning, sequencing, and clustering analysis. Some of the selected clones were peptide synthesized [18], analyzed for activity, and confirmed as CatE-activity-enhancing peptides. (f) The secondary library was generated using the primary library selection products followed by the peptide library with cDNA-tagged SF-link generation and selection process as described in steps 2–4.