Design of pH Sensitive Binding Proteins from the Hyperthermophilic Sso7d Scaffold

<div><p>We have engineered pH sensitive binding proteins for the Fc portion of human immunoglobulin G (hIgG) (hFc) using two different strategies – histidine scanning and random mutagenesis. We obtained an hFc-binding protein, Sso7d-hFc, through mutagenesis of the Sso7d protein from the hyperthermophilic archaeon <em>Sulfolobus solfataricus</em>; Sso7d-hFc was isolated from a combinatorial library of Sso7d mutants using yeast surface display. Subsequently, we identified a pH sensitive mutant, Sso7d-his-hFc, through systematic evaluation of Sso7d-hFc mutants containing single histidine substitutions. In parallel, we also developed a yeast display screening strategy to isolate a different pH sensitive hFc binder, Sso7d-ev-hFc, from a library of mutants obtained by random mutagenesis of a pool of hFc binders. In contrast to Sso7d-hFc, both Sso7d-his-hFc and Sso7d-ev-hFc have a higher binding affinity for hFc at pH 7.4 than at pH 4.5. The Sso7d-mutant hFc binders can be recombinantly expressed at high yield in <em>E. coli</em> and are monomeric in solution. They bind an epitope in the CH3 domain of hFc that has high sequence homology in all four hIgG isotypes (hIgG_1–4), and recognize hIgG_1–4as well as deglycosylated hIgG in western blotting assays. pH sensitive hFc binders are attractive candidates for use in chromatography, to achieve elution of IgG under milder pH conditions. However, the surface density of immobilized hFc binders, as well as the avidity effect arising from the multivalent interaction of dimeric hFc with the capture surface, influences the pH dependence of dissociation from the capture surface. Therefore, further studies are needed to evaluate if the Sso7d mutants identified in this study are indeed useful as affinity ligands in chromatography.</p> </div

Characterization of pH sensitivity for Sso7d-ev-hFc.

<p>(<b>A</b>) End-point assay to determine pH sensitivity for Sso7d-ev-hFc. Yeast cells displaying Sso7d-his-hFc were incubated with 2 µM hFc-biotin and the yeast-hFc complexes were dissociated in buffers at pH 7.4 and pH 4.5. Undissociated hFc remaining on yeast surface was detected using strep-PE. A cell sample where no dissociation step was carried out after hFc labeling at pH 7.4, and unstained cells were used as controls. (<b>C</b>) ELISA for determination of the apparent K_D of binding between hFc and Sso7d-ev-hFc, at pH 7.4 and pH 4.5. hIgG (2 µg/ml) was immobilized on a microtiter plate and incubated with twelve different concentrations of soluble Sso7d-ev-hFc. hIgG-bound Sso7d-ev-hFc was detected using an anti-his-alkaline phosphatase conjugated antibody, and p-nitrophenyl phosphate as the substrate. Error bars indicate the standard deviation of absorbance measurements at 405 nm.</p

Mutations in hFc leading to loss of binding to Sso7d-hFc.

<p>Amino acid changes in mutants with single amino acid substitutions that retain binding to Protein A but lose binding to Sso7d-hFc were selected as critical residues involved in the binding epitope of Sso7d-hFc on hFc.</p><p>(++) indicates wild type level binding;</p><p>(+) indicates binding somewhat reduced and.</p><p>(–) indicates loss of binding.</p

Epitope mapping of hFc binding Sso7d mutants.

<p>(<b>A</b>) Yeast surface displayed hFc was subjected to thermal denaturation at 99°C and subsequently detected using Sso7d-hFc or Protein A. Binding to Protein A is completely abolished, whereas binding to Sso7d-hFc is only slightly reduced. (<b>B</b>) Protein A and Sso7d-hFc can simultaneously detect the yeast-displayed hFc, confirming that they have distinct binding epitopes on hFc. (<b>C</b>) Individual hFc mutants from the final epitope mapping sorts are shown. These mutants lead to loss of binding of Sso7d-hFc to yeast displayed hFc (also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048928#pone-0048928-t004" target="_blank">Table 4</a>). (<b>D</b>) The mutations in (C) are mapped on the crystal structure of hFc. (<b>E</b>) Yeast surface displayed hFc was incubated with biotinylated Sso7d-hFc in the presence or absence of a high concentration of (unbiotinylated) Sso7d-ev-hFc. Cell surface bound hFc was detected using streptavidin-phycoerythrin (strep-PE). Sso7d-ev-hFc competes off the bound Sso7d-hFc confirming that Sso7d-ev-hFc binds an epitope that overlaps at least partially with that of Sso7d-hFc.</p

Characterization of pH sensitivity for Sso7d-hFc and strategies for engineering pH sensitivity.

<p>(<b>A</b>) End-point assay to determine pH sensitivity for Sso7d-hFc. Yeast cells displaying Sso7d-hFc were incubated with 100 nM hFc-biotin and the yeast-hFc complexes were dissociated in buffers at pH 7.4 and pH 4.5. Undissociated hFc remaining on yeast surface was detected using strep-PE. A cell sample where no dissociation step was carried out after hFc labeling at pH 7.4, and unstained cells were used as controls. (<b>B</b>) Dimeric hFc may form a multivalent association with cell surface displayed Sso7d-hFc. (<b>C</b>) ELISA for determination of the apparent K_D of binding between hFc and Sso7d-hFc, at pH 7.4 and pH 4.5. hIgG (1 µg/ml) was immobilized on a microtiter plate and incubated with twelve different concentrations of soluble Sso7d-hFc. hIgG-bound Sso7d-hFc was detected using an anti-his-alkaline phosphatase conjugated antibody, and p-nitrophenyl phosphate as the substrate. Error bars indicate the standard deviation of absorbance measurements at 405 nm. (<b>D</b>) Two different strategies used for engineering pH sensitive binding proteins are shown. The first strategy involves mutation of amino acid residues in the binding interface to histidine. The second strategy involves screening of pH sensitive binders from a library generated by random mutagenesis of hFc binders.</p

Biophysical characterization of hFc binding Sso7d mutants and western blotting analysis.

<p>(<b>A</b>) Size exclusion chromatography of Sso7d mutants purified by immobilized metal affinity chromatography (IMAC). The dashed box indicates elution peak for Sso7d mutants. Mutants were loaded on the column at a concentration of 2 mg/ml. Molecular weight estimates based on the retention time of Sso7d mutants in the column are consistent with the mutants being present in monomeric form. The other peak corresponds to a minor impurity with higher molar absorptivity than the Sso7d mutants (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048928#pone.0048928.s003" target="_blank">Figure S3</a>; SDS-PAGE analysis of fractions corresponding to the other peak do not show any detectable protein). (<b>B</b>) Circular dichroism spectra for Sso7d-hFc, Sso7d-his-hFc and Sso7d-ev-hFc at pH 7.4 and pH 4.5. The spectra at both pH values is essentially the same confirming that there is no change in secondary structure when the pH is lowered from 7.4 to 4.5 (<b>C</b>) Sso7d-hFc recognizes all four hIgG isotypes as well as the deglycosylated form of hIgG, when used as a primary reagent for detection in western blotting analysis. Lane 1: hIgG₁, lane 2: hIgG₂, lane 3: hIgG₃, lane 4: hIgG₄, lane 5: hIgG digested with PNGase F, lane 6: undigested hIgG (control). Similar results were observed with Sso7d-his-hFc and Sso7d-ev-hFc (data not shown).</p

Characterization of pH sensitivity for Sso7d-his-hFc.

<p>(<b>A</b>) End-point assay to determine pH sensitivity for Sso7d-his-hFc. Yeast cells displaying Sso7d-his-hFc were incubated with 100 nM hFc-biotin and the yeast-hFc complexes were dissociated in buffers at pH 7.4 and pH 4.5. Undissociated hFc remaining on yeast surface was detected using strep-PE. A cell sample where no dissociation step was carried out after hFc labeling at pH 7.4, and unstained cells were used as controls. (<b>B</b>) Histidine scanning analysis of Sso7d-hFc. Three out of ten histidine substitutions (L21H, M32H and I42H) lead to complete loss of binding to hFc, and therefore are involved in binding to hFc. Structure of the Sso7d scaffold with these residues in licorice representation is shown. This image was generated using Visual Molecular Dynamics (VMD) software. (<b>C</b>) ELISA for determination of the apparent K_D of binding between hFc and Sso7d-his-hFc, at pH 7.4 and pH 4.5. hIgG (2 µg/ml) was immobilized on a microtiter plate and incubated with twelve different concentrations of soluble Sso7d-his-hFc. hIgG-bound Sso7d-his-hFc was detected using an anti-his-alkaline phosphatase conjugated antibody, and p-nitrophenyl phosphate as the substrate. Error bars indicate the standard deviation of absorbance measurements at 405 nm. The inset shows binding curve at pH 4.5 over a wider range of Sso7d-his-hFc concentration.</p

Apparent equilibrium dissociation constants (K_D) for the Sso7d mutants.

<p>For each mutant, data from at least two independent experiments were fit globally to a four parameter logistic model using non-linear least squares regression for estimation of K_D. The corresponding 68% confidence intervals are shown in parentheses.</p>a<p>Not determined. The binding isotherm at pH 4.5 did not reach saturation even with 20–30 µM concentration of Sso7d-his-hFc and Sso7d-ev-hFc; consequently, an apparent K_D for the binding interaction at pH 4.5 could not be calculated.</p

Specificity of Sso7d-hFc, Sso7d-his-hFc and Sso7d-ev-hFc.

<p>(<b>A</b>) Yeast displayed Sso7d-hFc, Sso7d-his-hFc and Sso7d-ev-hFc were labeled with 1 µM hFc or 1 µM closely related non-human immunoglobulins, chicken IgY (cIgY), donkey IgG (dIgG), goat IgG (gIgG), mouse IgG (mIgG) and rabbit IgG (rIgG). Specific binding to hFc was observed. (<b>B</b>) Mutants were also labeled with hIgG fragments hFc, Fab and Fab2. Binding to Fab and Fab2 fragments was not observed.</p

pH sensitivity hFc binding to Sso7d mutants immobilized on a surface.

<p>(<b>A</b>) Sso7d-hFc, Sso7d-his-hFc and Sso7d-ev-hFc were recombinantly expressed with a C-terminal cysteine. 2 µg/ml of each mutant was chemically conjugated to wells of a maleimide-activated microtiter plate and incubated with 6 µM of hIgG-biotin. After a wash step, wells were incubated with buffers of pH 7.4, 4.5, 3.5 and 2.5 for 30 min with shaking, and the hIgG-biotin remaining undissociated was detected with alkaline phosphatase conjugated streptavidin and p-nitrophenyl-phosphate; absorbance measurements at 405 nm on a plate reader were obtained. Background-subtracted absorbance values were normalized by the absorbance value corresponding to dissociation in buffer at pH 7.4, and are reported. Error bars indicate standard deviation of measurements from six different wells. All three mutants show similar dissociation behavior in buffers at different pH. (<b>B</b>) Data from end-point assays for pH sensitivity of Sso7d mutants (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048928#pone-0048928-g001" target="_blank">Figures 1A</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048928#pone-0048928-g002" target="_blank">2A</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048928#pone-0048928-g004" target="_blank">4A</a>) are analyzed for comparison with (A). As in (A), Sso7d mutants immobilized on the surface of yeast are incubated with hFc-biotin and subsequently, the surface-bound hFc complexes are allowed to dissociate for 30 min in a buffer at pH 7.4 or pH 4.5; experiments with dissociation at pH 3.5 and pH 2.5 were not conducted. The fluorescence due to surface-bound hFc remaining undissociated was measured by flow cytometry, following labeling with strep-PE. Mean fluorescence values, normalized to the value for dissociation with pH 7.4 buffer, are plotted; data shown is the average from two separate experiments. A significantly greater fraction of hFc remains bound to Sso7d-hFc immobilized on the yeast cell surface.</p