Specific Six-Transmembrane Epithelial Antigen of the Prostate 1 Capture with Gellan Gum Microspheres: Design, Optimization and Integration

• Published in: International journal of molecular sciences Vol. 24; no. 3; p. 1949
• Main Authors: Batista-Silva, João, Gomes, Diana, Barroca-Ferreira, Jorge, Gallardo, Eugénia, Sousa, Ângela, Passarinha, Luís A
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 18-01-2023; MDPI
• Subjects: Affinity; Affinity chromatography; Androgens; Antigens; batch method; Bioreactors; Calcium; capture; Carbohydrates; Chemical elements; Chromatography; co-immunoprecipitation; Crosslinking; Design optimization; Gellan gum; Gellan gum microspheres; Humans; Immunoprecipitation; Immunotherapy; Ionic strength; Lysates; Male; Medical research; Membrane proteins; Microscopy; Microspheres; Morphology; Nickel; Polysaccharides, Bacterial - chemistry; Prostate; Prostate cancer; Protein purification; Proteins; Sodium chloride; STEAP1; batch method; capture; Gellan gum microspheres; co-immunoprecipitation; STEAP1
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms24031949

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
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% (
/
) 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.