Chemical and biological synthesis of an engineered affinity protein

• Autor(a) principal: Teixeira, Gonçalo Duarte Gomes
• Data de Publicação: 2017
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/26219
• topic: synthesis; expression; affinity; fusion protein; Domínio/Área Científica::Engenharia e Tecnologia::Engenharia Química

Protein engineering is na area of major interest for novel biotechnolgoical and biopharmaceutical applications. Several protein scaffolds have been explored as alternatives to antibodies and other affinity reagents. This work focuses on the detailed study of a previously selected protein domain for binding an important affinity ligand. This domain was produced chemically and biologically. The successfully produced protein by chemical synthesis was characterized through Mass Spectrometry and Circular Dichroism. To show the binding affinity for the target, several tests were performed, including binding tests after immobilization in a solid-support, MicroScale Thermophoresis (MST) and Multiparametric Surface Plasmon Resonance (MP-SPR). The biological production of the protein was performed by bacterial expression (in E. coli) and then purified. The expression of this domain was attempted alone or as a fusion protein with GFP. In the latter, BL21 Rosetta cells strain were used as expression host and the protein was purified by mixed-mode chromatography with the A4C7 synthetic ligand and by gel-filtration.