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A globotriaosylceramide (Gb3Cer) mimic peptide isolated from phage display library expressed strong neutralization to Shiga toxins |
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| Authors: | Yoshiko Miura Akio Sakaki Masamichi Kamihira Shinji Iijima Kazukiyo Kobayashi |
| Institution: | 1. Department of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University, Chikusa-ku, Nagoya 464-8603, Japan;2. Department of Biotechnology, Graduate School of Engineering, Nagoya University, Chikusa-ku, Nagoya 464-8603, Japan |
| Abstract: | A Gb3-trisaccharide mimic peptide was selected with biopanning from a phage display library against anti-Gb3 antibody to neutralize Shiga toxins (Stxs). Biopanning was carried out on a microplate immobilized with a Fab fragment of anti-Gb3 antibody and a subtraction procedure screening was applied to enhance specificity. The selected phage clones showed strong affinity to anti-Gb3 antibody and to Stxs. Among these clones, a 9-mer sequence WHWTWLSEY was determined as the strongest Gb3 mimic peptide and chemically synthesized. The peptide bound strongly to Stx-1 and Stx-2, though the binding was inhibited with Gb3Cer. Surface plasmon resonance (SPR) and fluorescent spectroscopy determined that the affinity of the peptide to both Stxs was strong. Neutralization activity was confirmed by in vitro assay with HeLa cells. The Gb3 mimic peptide potentially has great promise for use against Stxs. |
| Keywords: | ABTS 2&prime -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) BOP benzotriazole-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate BSA bovine serum albumin B simplicifolia Bandeiraea simplicifolia CBB Coomassie Brilliant Blue Cer ceramide Con A concanavalin A DMA N N-dimethylacetamide DMEM Dulbecco's modified Eagle's medium ELISA enzyme-linked immunosorbent assay FITC fluorescein isothiocyanote Fmoc fluorenylmethoxycarbonyl Gb3 globotriaosylceramide Gal(α1&ndash 4)Gal(β1&ndash 4)GlcβCer HBS-EP HEPES-buffered saline with EDTA and polysorbate added HEPES 4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid HOBt 1-hydroxybenzotriazole HPLC high-performance liquid chromatography HRP horseradish peroxidase Ig immunglobulin WST-8 2-(2-methoxy-5-nitrophenyl)-3-(4-nitrophenyl)-5-(2 4-disulfophenyl)-2H tetrazoluin monosodium salt MALDI matrix-assisted laser deposition ionization RCA120 Ricinus communis agglutinin 120 RMS root mean square SPR surface plasmon resonance Stx Shiga toxin TBS Tris-buffered saline TOF time of flight |
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