DARPin N3C and N2C libraries (Binz et al., 2004 (link)) were used and ribosome display (RD) selections were performed as described (Dreier and Plückthun, 2011 (link)). Green Fluorescent Protein (GFP) or mCherry, containing a C-terminal His-tag and an N-terminal avi-tag for in-vivo biotinylation during expression, were used as targets. Four rounds of RD with increasing stringency were carried out in solution with pull-down of the ternary complexes using streptavidin-coated magnetic beads. In the fourth round an off-rate selection with 300-fold excess of unbiotinylated GFP or mCherry over biotinylated target was performed. After the fourth round the enriched DNA pools were subcloned into the expression vector pQIq (Simon et al., 2012 (link)). For each selection, 192 single clones of each pool were screened by crude extract ELISA as described previously (Binz et al., 2004 (link); Zahnd et al., 2006 (link)). All single clones were also screened for binding to superfolder GFP (sfGFP). The nomenclature of the binders is as follows: the first number indicates the N2C or N3C library, respectively, the letters G or m indicate a DARPin specific for GFP or mCherry, respectively, and the last two- to three-digit number is a continuous numbering of the 192 clones that were screened. 3G86.1 and 3G86.32 come from the same initial clone that turned out to be a double transformant; single transformants were obtained by plasmid extraction and retransformation.
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