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Projeto de investigação
Viral Capture and Purification System: Smart macroporous structures for the affinity purification of retroviral particles
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Bioinspired and Sustainable Chitosan Based Monoliths for Antibody Capture and Release
Publication . Barroso, Telma; Roque, Ana Cecília Afonso; Ricardo, Ana Isabel Nobre Martins Aguiar de Oliveira; DQ - Departamento de Química; CQFB-REQUIMTE - Centro de Química Fina e Biotecnologia (Lab. Associado REQUIMTE); RSC - Royal Society of Chemistry
Chitosan-based monoliths activated by plasma technology induced the coupling of a robust biomimetic ligand, previously reported as an artificial Protein A, with high yields while minimizing the environmental impact of the procedure. Due to the high porosity, good mechanical and tunable physicochemical properties of the affinity chitosan-based monoliths, it is possible to achieve high binding capacities (150 ± 10 mg antibody per gram support), and to recover 90 ± 5% of the bound protein with 98% purity directly from cell-culture extracts. Therefore, the chitosan-based monoliths prepared by clean processes exhibit a remarkable performance for the one-step capture and recovery of pure antibodies or other biological molecules with biopharmaceutical relevance.
Exploring new protein-based scaffolds for bioengineering applications
Publication . Dias, Ana Margarida Gonçalves Carvalho; Roque, Ana; Casanova, Olga
The potential of WW domains as proteins scaffolds for the development of purification affinity reagents has been explored.
Minimal versions of native human Pin1 (hPin1_WW) and human YAP65 (hYAP65_WW) WW domains were produced through chemical synthesis using solid-phase peptide synthesis. After purification, characterization by mass spectrometry and circular dichroism demonstrated, respectively, the correct molecular mass, and the correct folding of WW domains with thermal stability comparable to the extended version expressed biologically. These peptides were further immobilized in chromatographic supports. Adsorbents bearing hPin1_WW captured phosphorylated peptides and proteins, whereas those modified with hYAP65_WW captured Proline-rich peptides. In both cases the binding and elution of the target peptides was achieved under mild conditions.
Encouraged by these results, a näive library based on the sequence of WW prototype was designed and generated. The library was further evolved in vitro through phage and ribosome display methodologies against Human Serum Albumin (HSA) and Immunoglobulin G (IgG). The phage display library leads to the identification of CW3S (a WW domain derived from Clone 3) as a potential HSA binder. This ligand was produced biologically in co-expression with GFP, and was also chemically synthesized. The affinity between CW3S and HSA was determined as Ka of 8.37x106 M-1 (KD=119nM) by ELISA. The chemically synthesized peptide was characterized by circular dichroism showing the folding and thermal stability similar to other native WW domains. This peptide was also immobilized in agarose and captured HSA (0.291μg
protein/mg support at 4°C).
This work strongly demonstrated the robustness of the WW domains to withstand
modifications and mutations, therefore possessing a clear potential for application as a protein scaffold.
Dextran-Coated Magnetic Supports Modified with a Biomimetic Ligand for IgG Purification
Publication . Santana, Sara D. F.; Dhadge, Vijaykumar L; Roque, Ana Cecília Afonso; CQFB-REQUIMTE - Centro de Química Fina e Biotecnologia (Lab. Associado REQUIMTE); DQ - Departamento de Química; ACS - American Chemical Society
Dextran-coated iron oxide magnetic particles modified with ligand 22/8, a protein A mimetic ligand, were prepared and assessed for IgG purification. Dextran was chosen as the agent to modify the surface of magnetic particles by presenting a negligible level of nonspecific adsorption. For the functionalization of the particles with the affinity ligand toward antibodies, three methods have been explored. The optimum coupling method yielded a theoretical maximum capacity for human IgG calculated as 568 ± 33 mg/g and a binding affinity constant of 7.7 × 10⁴ M⁻¹. Regeneration, recycle and reuse of particles was also highly successful for five cycles with minor loss of capacity. Moreover, this support presented specificity and effectiveness for IgG adsorption and elution at pH 11 directly from crude extracts with a final purity of 95% in the eluted fraction.
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Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
3599-PPCDT
Número da atribuição
PTDC/EBB-BIO/118317/2010