Single Domain Antibodies as a Powerful Tool for High Quality Surface Plasmon Resonance Studies

<div><p>Single domain antibodies are recombinantly expressed functional antibodies devoid of light chains. These binding elements are derived from heavy chain antibodies found in camelids and offer several distinctive properties for applications in biotechnology such as small size, stability, solubility, and expression in high yields. In this study we demonstrated the potential of using single domain antibodies as capturing molecules in biosensing applications. Single domain antibodies raised against green fluorescent protein were anchored onto biosensor surfaces by using several immobilization strategies based on Ni²⁺:nitrilotriacetic acid-polyhistidine tag, antibody-antigen, biotin-streptavidin interactions and amine-coupling chemistry. The interaction with the specific target of the single domain antibodies was characterized by surface plasmon resonance. The immobilized single domain antibodies show high affinities for their antigens with K_D = 3–6 nM and outperform other antibody partners as capturing molecules facilitating also the data analysis. Furthermore they offer high resistance and stability to a wide range of denaturing agents. These unique biophysical properties and the production of novel single domain antibodies against affinity tags make them particularly attractive for use in biosensing and diagnostic assays.</p></div

Interaction of GFP with a CM5 surface functionalized with GFP-Nb.

<p>Depicted sensorgrams were obtained for GFP-Nb concentration of 0.2, 0.5, 1, 2, 5, 7, 10, 15, 20, 50, 100, 200, 500, 1000 nM (from bottom to top curve).</p

Endpoint response of the reference subtracted binding curves describing the interaction between the GFP-Nb and the different surfaces as function of GFP-Nb concentration.

<p>The red, blue and black squares are the raw data for the Ni:NTA, the anti-polyhistidine and the CAP chip respectively, and the lines are the best fit of the data set to the Hill’s equation (R² = 0.99). As choosing a Hill’s coefficient equal to 1 did not produce a good fit of the data describing the interaction between the GFP-Nb and the anti-polyhistidine and the CAP-chip (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124303#pone.0124303.s002" target="_blank">S2 Fig</a>), a Hill’s coefficient of 2 was set for these lines.</p

GFP:GFP-Nb complex.

<p>Binding of GFP to GFP-Nb immobilized on Ni:NTA (A), anti-polyhistidine antibody on CM5 (B) and streptavidin on CAP chip (C). Black lines are raw data and the red lines are the fitting to a 1:1 binding model. Depicted sensorgrams were obtained for GFP concentration of 0.1, 0.5, 1, 5, 10, 50, 100, 200, 500, 1000 nM (from bottom to top curve).</p

GST-pull down experiments with radiolabelled GASP-1, -2, -3, -6, -7 and -9 and GST-fused receptor C-tails.

<p><i>A,</i> GASP-1, -2 and -3 showed medium to strong interactions with some GPCR C-tails but no interaction was detected with the two one-transmembrane receptor C-tails (TGF_β and IGF₁). <i>B,</i> GASP-7 showed weak to medium interactions with some GPCR C-tails. GASP-6 and -9 showed very weak interactions with all tested receptors. No interaction was detected with TGF_β and IGF C-tails. Data were quantified by Phosphor-imaging. Results are shown as percent of the [³⁵S]-GASPs input retained by the GST-fused receptor C-tails and correspond to the mean ± S.E.M of three independent experiments. Lower panels correspond to representative gel images. 5HT₇, 5-hydroxytryptamine 7 receptor; ADRB1, β₁ adrenergic receptor; CALCR, calcitonin receptor; DOR, δ-opioid receptor; FZ₄, frizzled 4 receptor; H₂, histamine 2 receptor; IGF₁, insulin growth factor I receptor; KOR, κ-opioid receptor; M₁, muscarinic M₁ acetylcholine receptor; M₂, muscarinic M₂ acetylcholine receptor; MOR, µ-opioid receptor; ORL₁, opioid receptor-like 1; TXA₂, α isoform of the thromboxane A₂ receptor; TGF_β, type III transforming growth factor β receptor.</p

The GASP motif is critical for the interaction of GASP-2 with GPCRs.

<p><i>A,</i> GST-pull down experiments with two truncated mutants of GASP-2 and ADRB1, M₁ and CALCR C-tails. Grey boxes represent the 15 AA GASP motifs. Deletion analysis revealed that the central domain of GASP-2, which contains the two GASP motifs, is critical for the interaction between GASP-2 and ADRB1, M₁ and CALCR C-tails. <i>B,</i> GST-pull down experiments with full-length GASP-2 where one (GASP2-m1 and GASP2-m2) or both GASP motifs (GASP2-dm) were mutated. Grey boxes represent the wild-type motifs and X represent the mutant motifs. Consensus sequences are given for wild-type and mutant motifs. Mutated amino acids are underlined. Site directed mutagenesis analysis of these two repeated motifs showed that they played a crucial role in the interaction of GASP-2 with the three receptor C-tails tested here. Results are shown as percent of the wild-type GASP-2 interaction and correspond to the mean ± S.E.M of three independent experiments.</p

A small synthetic peptide derived from the GASP motif of GASP-2 blocks the interaction between GASPs and GPCR C-tails in GST-pull down experiments.

<p>A, GASP peptide competes for the interaction between GASP-2 and GST-fused ADRB1 C-tail. The scrambled peptide displayed no significant effect on the interaction between GASP-2 and ADRB1. <i>B,</i> A fixed concentration of GASP peptide (150 µM) inhibits the interaction between GASP-1, -2 or -7 with ADRB1 C-tail, but not the scrambled peptide. <i>C,</i> Phosphor-imaging quantification of the competition experiments for the interaction between GASP-1, -2 and -7 and four different receptor C-tails with GASP peptide. A fixed concentration of GASP peptide (150 µM) strongly inhibited interactions of GASPs with ADRB1, M₁, CALCR and TXA₂ C-tails. Results are represented as percent of the interaction between the corresponding GASPs and GPCRs in absence of peptide (mean ± S.E.M of three independent experiments).</p

Schematic comparison of GASP family members.

<p>Black boxes represent the conserved carboxyl-terminal domain of 250 amino acids. The percentage of identical amino acids shared with GASP-1 is indicated within each box. Small grey boxes represent a highly conserved motif of 15 amino acids that is repeated 22 times in GASP-1 and two times in GASP-2 to -5. The consensus sequence of this motif is: (E/D/G) (E/D) E X (I/L/V/S/T) (I/V/A/F) (G/N) (S/T) W F W (A/V/T/S/D/E) (G/E/R) (E/D/K) (E/D/K/A/Q). For GASP-2, two regions showing significant sequence homology with GASP-1 are separated by a gap represented by dotted lines. GASPs accession numbers from SPtrEMBL database are indicated on the left of the figure.</p

The central domain of GASP-1 co-immunoprecipitates with GPCRs in cells.

<p>The central domain of GASP-1 (amino-acids 380 to 1073 of GASP-1 in pcDNA3.1) was transiently transfected in HEK293 cells stably expressing GFP-tagged ADRB1, ADRB2, CALCR or M₁ receptor. HEK293 cells stably expressing MyrPalm-mYFP and transiently transfected with the central domain of GASP-1 were used as a negative control. The central domain of GASP-1 co-immunoprecipitated with the four different GPCRs while no co-immunoprecipation was observed in cells expressing the central domain of GASP-1 alone or co-expressing this domain with myristoylated-palmitoylated mYFP (MyrPalm-mYFP).</p

Purified full-length GPCRs dose-dependently bind to the central domain of GASP-1 in SPR experiments.

<p><i>A</i>, Interaction of the central domain of GASP-1 compared to the full-length protein with GST-fused ADRB2 and CNR2 C-tails by GST pull down experiments. The results show that both receptors interact <i>in vitro</i> with GASP-1 and that the central part of GASP-1 is strongly involved in the interaction with ADRB2 and CNR2. <i>B</i>, Binding of a range of concentrations of ADRB2 to the central domain of GASP-1. <i>C</i>, Binding of a range of concentrations of CNR2 to the central domain of GASP-1. Overall, we observed a dose-dependent binding of ADRB2 and CNR2 with the central domain of GASP-1. The receptor concentrations are indicated on the figures. All curves are double referenced and corrected for changes in capture density of the central domain of GASP-1. ADRB2, β₂ adrenergic receptor; CNR2, cannabinoid receptor type 2.</p