title: Tryptophan tags and de novo designed complementary affinity ligands for the expression and purification of recombinant proteins.
abstract: A common strategy for the production and purification of recombinant proteins is to fuse a tag to the protein terminal residues and employ a "tag-specific" ligand for fusion protein capture and purification. In this work, we explored the effect of two tryptophan-based tags, NWNWNW and WFWFWF, on the expression and purification of Green Fluorescence Protein (GFP) used as a model fusion protein. The titers obtained with the expression of these fusion proteins in soluble form were 0.11mgml-1and 0.48mgml-1for WFWFWF and NWNWNW, respectively. A combinatorial library comprising 64 ligands based on the Ugi reaction was prepared and screened for binding GFP-tagged and non-tagged proteins. Complementary ligands A2C2 and A3C1 were selected for the effective capture of NWNWNW and WFWFWF tagged proteins, respectively, in soluble forms. These affinity pairs displayed 106M-1affinity constants and Qmax values of 19.11±2.60ugg-1and 79.39ugg-1for the systems WFWFWF AND NWNWNW, respectively. GFP fused to the WFWFWF affinity tag was also produced as inclusion bodies, and a refolding-on column strategy was explored using the ligand A4C8, selected from the combinatorial library of ligands but in presence of denaturant agents.
authors: UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal; IBET - Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal; Institute of Biotechnology, Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, CB2 1QT, Cambridge, UK.
authors: Ana Sofia
authors: AS
authors: Pina
authors: UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.
authors: Sara
authors: S
authors: Carvalho
authors: UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal; IBET - Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal.
authors: Ana Margarida G C
authors: AM
authors: Dias
authors: UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.
authors: Márcia
authors: M
authors: Guilherme
authors: UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.
authors: Alice S
authors: AS
authors: Pereira
authors: UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.
authors: Luciana T
authors: LT
authors: Caraça
authors: IBET - Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal.
authors: Ana Sofia
authors: AS
authors: Coroadinha
authors: Institute of Biotechnology, Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, CB2 1QT, Cambridge, UK.
authors: Christopher R
authors: CR
authors: Lowe
authors: UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal. Electronic address: cecilia.roque@fct.unl.pt.
authors: A Cecília A
authors: AC
authors: Roque
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D002479:Inclusion Bodies / Q000378:metabolism; D008024:Ligands; D011489:Protein Denaturation; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D012995:Solubility; D014364:Tryptophan / Q000737:chemistry* / Q000302:isolation & purification*
chemical_list: D008024:Ligands; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D014364:Tryptophan
keywords: Affinity chromatography; Affinity ligand; Affinity tag; Combinatorial chemistry; Green fluorescent protein; Recombinant proteins; Ugi reaction