IMAC Purification of scFv TEG4-2c produced and secreted by <i>P</i>. <i>pastoris</i>.

<p>Data are standardized for 1 L culture media; the values are the mean of 7 independent experiments ± SD values.</p

pPICZαA expression vector according to the EasySelect <i>Pichia</i> Expression Kit Manual (Invitrogen) and a schematic representation of TEG4-2c scFv with the protein sequence.

<p><b>(A)</b>: All the featured restriction sites are unique. 5´ AOX1: promoter region of AOX1; TT AOX1: transcription termination of AOX1; PTEF1: promoter of TEF1; PEM7: promoter of EM7; Zeocin resistance: Sh ble ORF; CYC1 TT: transcription termination of CYC1. <b>(B)</b>: The TEG4-2c scFv coding sequence was cloned between <i>Pml</i>I and <i>Xba</i>I sites and the protein sequence of recombinant tag-scFv including the 6His-tag and the 2 cysteine are highlighted in green. The α-factor signal sequence is represented in blue and the C-myc tag is highlighted in yellow.</p

Comparison of the immunoreactivity of TEG4-2c scFv to atherosclerotic tissues of different species by IHC analysis and ELISA assays.

<p><b>A</b> (<b>a-r)</b>: IHC assays on atherosclerotic tissues: similarly to positive controls e.g; anti-CD41 (anti-αIIb) (e), RAM11 and PGM1 (anti-CD68 antibodies targeting rabbit and human macrophages respectively) (k, q) and AP2 (anti-αIIbβ3 antibody) (a; g; m), TEG4-2c scFv specifically recognizes the injured areas of the aorta sections from different species (c; i; o). Binding of antibodies was visualized via HRP-anti-6His (scFv); HRP anti-rabbit IgG (CD41) and HRP anti-mouse IgG (RAM11, AP2). Negative controls were secondary antibody only (b; d; f; h; j; l; n; p; r). Nuclei were counterstained with hematoxylin <b>B</b>: ELISA tests on atheromatous and healthy aorta proteins: TEG4-2c shows a better recognition of atheromatous proteins. RAM11 and AP2 were used as positive controls. Negative controls were secondary antibody only. Binding of antibodies was visualized via HRP-6His or HRP-anti-mouse IgG. OD value represents absorbance at 450 nm. Values represent mean (n = 3) ± SD (error bars materialized the SD)</p

A Recombinant Human Anti-Platelet scFv Antibody Produced in <i>Pichia pastoris</i> for Atheroma Targeting - Fig 3

<p><b>Purification of recombinant TEG4-2c scFv A: IMAC chromatogram</b>. The HisTrap Excel resin (5 mL) was equilibrated with 50 mM Tris-HCl pH 7.5, 500 mM NaCl (buffer A at a flow rate of 3 mL/min). <i>Pichia pastoris</i> expression broth supernatant containing the TEG4-2c scFv was injected into the column. The column was then washed with buffer A until absorbance at 280nm reached the baseline. (Dot Line): The elution was carried out in two steps using 5% and 30% buffer B (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 500 mM imidazole) corresponding respectively to 25 mM and 150 mM imidazole. <b>B: Electrophoretic analysis of one step IMAC purification of recombinant TEG4-2c scFv</b>. 12% SDS-PAGE stained with colloidal blue, MW: molecular weight ladder (KDa). [S]: 5x concentrated culture supernatant. [TF]: 5x concentrated flow-through. W: 25 mM imidazole washing fraction. E: 150 mM elution fraction. E_PBS: Elution fraction dialyzed against PBS.</p

Binding of scFv TEG4-2c to αIIbβ3 by SPR and to whole platelets by BLI.

<p><b>A: SPR sensorgrams</b>. The ligand αIIbβ3 was immobilized on CM5 chip by amine coupling with a density of 8000 RU. Serial dilutions of TEG4-2c in HBS-EP running buffer were injected over the ligand corresponding to 94, 188 and 390 nM. Sensorgrams show a binding concentration-dependent of TEG4-2c scFv. <b>B: BLI analysis</b>. TEG4-2c scFv (ligand) was loaded on HIS2 biosensor (anti-penta Histidine Ab) at 21 μg/mL. Whole platelets (analyte) concentrations converted into αIIbβ3 molarities were: 41.5, 8.3, 4.15, 0.83, 0.415, 0.083 and 0.0415 nM. Additionally one sensor pair was used to record the buffer reference signals. TEG4-2c scFv reacts with αIIbβ3 in its natural conformation in a concentration dependent manner.</p

TEG4-2c scFv production process. A: Fed batch fermentation history plot.

<p>Stirring, pO₂ and OD₆₀₀ values are plotted versus time during the cultivation of <i>P</i>. <i>pastoris</i> in BMGY medium. Cultures were induced with methanol at t = 0 (24 h after starting the batch phase) during the fed batch phase the methanol was added every 12 h or 6h (black arrows) to a final concentration of 0.6%. The average values are shown with error bars representing the standard deviation (n = 5). 1 OD₆₀₀ unit was equivalent to 0.29 mg/mL dry weight. <b>B: Dot-blot analysis of supernatants from recombinant <i>P</i>. <i>pastoris</i> culture</b>. Fifty microliters samples from non-induced culture (NI) and from day 1 to day 5 induced cultures (I1d to I5d) were undiluted (a) or diluted (b = 1:10; c = 1:50) and blotted on a nitrocellulose membrane. The recombinant TEG4-2c scFv were detected with the Anti-6His antibody and revealed by colorimetric analysis.</p

Binding assessment of TEG4-2c scFv to human platelets by flow cytometry and ELISA tests.

<p><b>A</b>: Binding of TEG4-2c scFv on thrombin-activated human (A-PL) or non-activated—platelets (NA-PL) analysed by flow cytometry. PAC-1 IgM murine antibody serves as a positive control. Binding of antibody to the platelets was further detected by incubation with Alexa Fluor 488 anti-6His or anti-mouse IgM antibodies. Negative controls were secondary antibody only. Histograms depict representative data ± SD of three independent experiments. Quantitative fluorescence intensities (in Geo mean) are stated under each respective histogram. <b>B</b>: Binding of TEG4-2c scFv on TRAP-activated-human (+ TRAP) or non-activated platelets (-TRAP) analysed by flow cytometry. Quantitative fluorescence intensities (in Geo mean) are stated under each respective histogram. <b>C</b>: Representative whole cell (A-PL, NA-PL) and purified proteins (αIIbβ3, BSA) ELISA with TEG4-2c scFv. A murine anti-αIIbβ3 antibody AP2 was used as positive control. Negative controls were secondary antibody only. Binding of antibodies was visualized via HRP-6His or HRP-anti-mouse IgG. OD value represents absorbance at 450 nm. Plots represent the mean values ± SD (n = 3)</p