Batista-Silva, JoãoGomes, DianaBarroca-Ferreira, JorgeGallardo, EugéniaSousa, ÂngelaPassarinha, Luís A.2023-07-042023-07-042023-01-181661-6596PURE: 65198713PURE UUID: 590a37f1-7cc6-460d-a531-7d93bc57e814Scopus: 85147895711WOS: 000929578200001PubMed: 36768273PubMedCentral: PMC9916199http://hdl.handle.net/10362/154840Funding 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.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.193102676engbatch methodcaptureco-immunoprecipitationGellan gum microspheresSTEAP1CatalysisMolecular BiologySpectroscopyComputer Science ApplicationsPhysical and Theoretical ChemistryOrganic ChemistryInorganic Chemistryjournal article10.3390/ijms24031949https://www.scopus.com/pages/publications/85147895711