Figure 1. Selection of sybodies against membrane proteins within three weeks.
(A) Three synthetic libraries exhibiting highly variable randomized surfaces (concave, loop and convex) each harboring a diversity of 9 × 1012 were designed based on thermostabilized nanobody frameworks. CDR1, CDR2 and CDR3 are colored in yellow, orange and red, respectively. (B) The in vitro selection platform is built as a selection cascade, starting with 1012 sybodies displayed on ribosomes for pre-enrichment, followed by a focused phage display library of 107 clones and binder identification by ELISA (typically 96 clones). The platform builds on fragment exchange (FX) cloning using Type IIS restriction sites encoded on the phage display (pDX_init) and expression vector (pSb_init) backbones, which generate AGT and GCA sticky ends for PCR-free subcloning. Key elements for reliable selections against membrane proteins are the shape variability of the sybody libraries, exceptionally high experimental diversities using ribosome display and the change of display system during the selection process.
Figure 1—figure supplement 1. Variable sybody scaffolds based on three camelid nanobodies.
CDR1, CDR2 and CDR3 are colored in yellow, orange and red, respectively. In the left panel, crystal structures of camlid nanobodies in complex with GFP (PDB: 3K1K) (A), a GPCR (PDB: 3P0G) (B) and Lysozyme (PDB: 1ZVH) (C) are shown, which served as starting point to delineate scaffolds for randomization. Nanobody residues contacting the target proteins are depicted as sticks. The target proteins are colored in blue. In the middle panel, homology models of three framework nanobodies are shown as cartoons and randomized residues (defined as serines and threonines in these examples) are highlighted as sticks. The three sybody libraries exhibit a concave (A), loop (B) or convex (C) binding surface, respectively. The right panel shows the randomized surface of the three libraries with the side chains of the randomized positions highlighted in color. Note that the concave library contains randomized residues outside of the CDR regions, which are colored in purple.
Figure 1—figure supplement 2. Framework sequences and randomized positions.
(A and B) Sequences of the framework sybodies are aligned with the sequences of their natural precursors. The frameworks of the concave and the loop library are identical (A) while the convex library has its own scaffold (B). Residues of the natural precursor nanobodies differing from the framework sequence are marked in blue. The three CDR regions are underlined. Invariant CDR residues contributing to the hydrophobic core of the respective scaffold are marked in green. Note that the differently shaped libraries exhibit alternative sets of invariant CDR residues that precisely match the corresponding scaffolds. This harmonization is a critical and unprecedented feature of our synthetic nanobdy libraries, as it allows for the first time to include variable CDR lengths without the risk of scaffold destabilization. Randomized residues are highlighted as red S (for which randomization mixture one was used), as red T (mix 2) and orange T (mix 3). (C) Amino acid composition of randomized positions obtained by three different trinucleotide randomization mixtures. The rationale behind the three randomization mixtures is provided in the main text.
Figure 1—figure supplement 3. Biophysical characterization of sybodies.
Three framework sybodies representing the concave, the loop and the convex library and containing serines and threonines in the randomized positions were generated by gene synthesis (sequences provided in Figure 1—figure supplement 2). (A) SEC analysis of periplasmatically expressed concave, loop and convex framework sybodies using a Superdex 75 300/10 GL column. (B) Determination of melting temperature (Tm) of framework sybodies and their natural precursors 3K1K and 1ZVH using dye SYPRO Orange (ThermoFluor). Representative data of two technical replicates are shown.
Figure 1—figure supplement 4. Ribosome display of single domain antibodies.
(A) The non-randomized convex sybody was either purified containing a C-terminal 3x-FLAG tag or displayed on ribosomes containing the same tag using the commercial kit PUREfrexSS (GeneFrontier). 3C protease cleavage was used to liberate the displayed sybody from the ribosomal complex. Western blotting analysis using anti-3x-FLAG antibody and purified sybody as standard revealed a display efficiency of 82% of input mRNA for ribosome display. (B) 106 mRNA molecules encoding the GFP-specific 3K1K nanobody were displayed on ribosomes using PUREfrexSS together with 1012 mRNA molecules encoding the non-randomized convex sybody. The ribosomal complexes were pulled down using either biotinylated GFP or MBP immobilized on magnetic beads. The mRNA of isolated ribosomal complexes was isolated, reverse transcribed and the resulting cDNA was analyzed by qPCR performing technical triplicates. This analysis revealed that 84.6 ± 3.5% (error corresponds to standard deviation) of the input 3K1K mRNA was retrieved on GFP-coated beads, while virtually no background binding of the non-randomized convex sybody nor 3K1K binding to MBP was observed.
Figure 1—figure supplement 5. FX cloning vector series for phage display and purification of sybodies and nanobodies.
Sybody pools from ribosome display (or nanobodies from immunized camelids) are amplified with primers containing restriction sites of Type IIS enzyme BspQI (isoschizomer of SapI) to generate AGT and GCA overhangs. BspQI restriction sites generating the same overhangs were introduced into the backbones of vector pDX_init for phage display and pSb_init for periplasmatic expression and attachment of Myc- and His-tag. Note that in pDX_init and pSb_init the BspQI restriction sites are part of the sybody open reading frame. Finally, sybodies/nanobodies are sub-cloned from pSb_init to the destiny vectors pBXNPH3 or pBXNPHM3 for periplasmic expression. Tag-less sybodies/nanobodies for structural biology purposes can be obtained by 3C protease cleavage. Importantly, the vector series permits for PCR-free subcloning once the sybodies have been inserted into phage display vector pDX_init. The vectors were made available through Addgene (for Addgene IDs, see Table 3).
Figure 1—figure supplement 6. Improvement of the sybody selection procedure.
(A) Three rounds of ribosome display using the same type of magnetic beads for target immobilization (Dynabeads Myone Streptavidin T1) failed to generate sybodies against ABC transporter TM287/288. Pool enrichment against TM287/288 compared to negative control AcrB was poor. No positive ELISA hits were identified. (B) Sybody selections against TM287/288 were performed applying one round of ribosome display followed by two rounds of phage display using Dynabeads Myone Streptavidin T1 for target immobilization. The pool was enriched approximately 30 fold and a few positive ELISA hits were found. Purification of identified sybodies failed. (C) Sybody selections against ABC transporter IrtAB, a homologue of TM287/288 sharing a sequence identity of 27%, was performed as in (B), but using different immobilization chemistries (Dynabeads Myone Streptavidin T1 for ribosome display, Maxisorp microtiter plates for the first phage display round and Dynabeads Myone Streptavidin C1 for the second phage display round) to suppress accumulation of background binders. Strong enrichment was observed and a high number of positive ELISA hits were identified. Only 27% of positive ELISA hits were unique sybodies with moderate affinities. (D) Final optimized sybody selection protocol as described in the materials and methods section. Diversity bottlenecks were removed by using Taq DNA polymerase for cDNA amplification and increasing the working volume of the first phage display round. An off-rate selection step was introduced in the second phage display round. Enrichment and number of ELISA hits was similar to the selection shown in (C). The number of unique ELISA hits increased to 83% and high affinity binders were obtained. The binders obtained in (D) against TM287/288 are described in detail in main Figures 3 and 4.