Design, construction, and characterization of a second‐generation DARPin library with reduced hydrophobicity

Abstract

Designed ankyrin repeat proteins (DARPins) are well-established binding molecules based on a highly stable nonantibody scaffold. Building on 13 crystal structures of DARPin-target complexes and stability measurements of DARPin mutants, we have generated a new DARPin library containing an extended randomized surface. To counteract the enrichment of unspecific hydrophobic binders during selections against difficult targets containing hydrophobic surfaces such as membrane proteins, the frequency of apolar residues at diversified positions was drastically reduced and substituted by an increased number of tyrosines. Ribosome display selections against two human caspases and membrane transporter AcrB yielded highly enriched pools of unique and strong DARPin binders which were mainly monomeric. We noted a prominent enrichment of tryptophan residues during binder selections. A crystal structure of a representative of this library in complex with caspase-7 visualizes the key roles of both tryptophans and tyrosines in providing target contacts. These aromatic and polar side chains thus substitute the apolar residues valine, leucine, isoleucine, methionine, and phenylalanine of the original DARPins. Our work describes biophysical and structural analyses required to extend existing binder scaffolds and simplifies an existing protocol for the assembly of highly diverse synthetic binder libraries.

Altering lamina assembly identifies lamina-dependent and -independent functions for A-type lamins

“…The selection of DARPins that specifically bind to lamin A was performed with standard ribosome display using an N 3 C library (Seeger et al, 2013). As the target protein, we used recombinant human lamin A that had been reconstituted in dimerization buffer (Taimen et al, 2009).…”

“…Ribosome display, crude extract ELISA and surface plasmon resonance analysis For selection of lamin-A-specific DARPins, an N 3 C library was used, and four standard ribosome-display selection rounds (Seeger et al, 2013) were performed against immobilized 6His-TEV laminA Avi , reconstituted in a 'dimerization buffer' containing 300 mM NaCl, 25 mM Tris ( pH 8.0), 2 mM EDTA and 1 mM DTT Taimen et al, 2009). Crude extract ELISA against reconstituted 6His-TEV laminA Avi and surface plasmon resonance analysis on a Proteon XPR36TM (Bio-Rad Laboratories, Inc.) was performed as described previously (Seeger et al, 2013).…”

“…Crude extract ELISA against reconstituted 6His-TEV laminA Avi and surface plasmon resonance analysis on a Proteon XPR36TM (Bio-Rad Laboratories, Inc.) was performed as described previously (Seeger et al, 2013).…”

“…6His DARPins (∼18 kDa) and 6His-TEV-mCherry DARPins (∼45 kDa) were expressed in BL21-CodonPlus(DE3) grown in terrific broth after induction with 1 mM IPTG for approximately 5 h at 37°C and 16 h at 18°C, respectively, and purified as previously described (Seeger et al, 2013).…”

Consensus design of a NOD receptor leucine rich repeat domain with binding affinity for a muramyl dipeptide, a bacterial cell wall fragment

“…Many repeat proteins, such as ankyrin repeats (ANK), tetratricopeptide repeats (TPR), leucine rich repeats (LRR), and armadillo (ARM) repeats have been successfully used for the development of novel binding scaffolds. 5,[14][15][16][17][18][19][20] The evolutionary advantage of a modular architecture is the possibility to evolve the function Additional Supporting Information may be found in the online version of this article.…”