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http://hdl.handle.net/10362/154840
Título: | Specific Six-Transmembrane Epithelial Antigen of the Prostate 1 Capture with Gellan Gum Microspheres |
Autor: | Batista-Silva, João Gomes, Diana Barroca-Ferreira, Jorge Gallardo, Eugénia Sousa, Ângela Passarinha, Luís A. |
Palavras-chave: | batch method capture co-immunoprecipitation Gellan gum microspheres STEAP1 Catalysis Molecular Biology Spectroscopy Computer Science Applications Physical and Theoretical Chemistry Organic Chemistry Inorganic Chemistry |
Data: | 18-Jan-2023 |
Resumo: | This work demonstrates the potential of calcium- and nickel-crosslinked Gellan Gum (GG) microspheres to capture the Six-Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) directly from complex Komagataella pastoris mini-bioreactor lysates in a batch method. Calcium-crosslinked microspheres were applied in an ionic exchange strategy, by manipulation of pH and ionic strength, whereas nickel-crosslinked microspheres were applied in an affinity strategy, mirroring a standard immobilized metal affinity chromatography. Both formulations presented small diameters, with appreciable crosslinker content, but calcium-crosslinked microspheres were far smoother. The most promising results were obtained for the ionic strategy, wherein calcium-crosslinked GG microspheres were able to completely bind 0.1% (v/v) DM solubilized STEAP1 in lysate samples (~7 mg/mL). The target protein was eluted in a complexed state at pH 11 with 500 mM NaCl in 10 mM Tris buffer, in a single step with minimal losses. Coupling the batch clarified sample with a co-immunoprecipitation polishing step yields a sample of monomeric STEAP1 with a high degree of purity. For the first time, we demonstrate the potential of a gellan batch method to function as a clarification and primary capture method towards STEAP1, a membrane protein, simplifying and reducing the costs of standard purification workflows. |
Descrição: | Funding Information: The authors acknowledge the support from FEDER funds through the POCI-COMPETE 2020–Operational Programme Competitiveness and Internationalisation in Axis I–Strengthening Research, Technological Development and Innovation (Project POCI-01-0145-FEDER-007491).This work was also supported by the Associate Laboratory Institute for Health and Bioeconomy–i4HB (project LA/P/0140/2020) which are financed by National Funds from FCT/MCTES. Publisher Copyright: © 2023 by the authors. |
Peer review: | yes |
URI: | http://hdl.handle.net/10362/154840 |
DOI: | https://doi.org/10.3390/ijms24031949 |
ISSN: | 1661-6596 |
Aparece nas colecções: | Home collection (FCT) |
Ficheiros deste registo:
Ficheiro | Descrição | Tamanho | Formato | |
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Specific_Six_Transmembrane.pdf | 3,03 MB | Adobe PDF | Ver/Abrir |
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