Development of biological and synthetic affinity ligands for human serum albumin

• Autor(a) principal: Viecinski, Aline Canani
• Data de Publicação: 2015
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/61271
• topic: Human serum albumin; biological ligands; WW domain; synthetic; ligand; combinatorial chemistry; Domínio/Área Científica::Engenharia e Tecnologia::Engenharia Química

This work aimed the development of biological and synthetic affinity ligands for human serum albumin (HSA). The first approach was to optimize the expression of a biological ligand, named WW Clone 3, which was previously selected from a phage display library using HSA as a target. The expression of WW Clone 3 in Escherichia coli strain BL21 (DE3) was attempted at three different temperatures (18ºC, 25ºC and 30ºC), with 1mM Isopropyl-β-D-thiogalactopyranoside (IPTG). The best temperature for cell growth was 30ºC, producing a larger amount of total protein with approximately 45 mg/ml in the soluble fraction and 10mg/ml in the insoluble fraction. However, WW Clone 3 was difficult to produce. The second approach was to develop synthetic affinity ligands to HSA based on solid-phase synthesis of combinatorial libraries. The libraries were designed on the basis of amino acids from protein PAB (Peptostreptococcal albumin-binding) and drugs that bind naturally to the domain II of HSA. Two libraries were synthesized through the Ugi and Triazine reaction with 88 and 64 ligands, respectively. The libraries were screened for binding to pure HSA and pure immunoglobulin G (IgG), to assess the selectivity towards HSA. The ligands that had the highest affinity for HSA and lowest affinity for IgG were re-screened to confirm the results. Two ligands, A3A2 and A6A5 from the Triazine library appeared as the most selective for HSA and therefore more promising for the capture of this protein.