@article {Pina2016,
	title = {Tryptophan tags and de novo designed complementary affinity ligands for the expression and purification of recombinant proteins},
	journal = {Journal of Chromatography A},
	volume = {1472},
	year = {2016},
	note = {<p>n/a</p>
},
	pages = {55{\textendash}65},
	abstract = {<p>A common strategy for the production and purification of recombinant proteins is to fuse a tag to the protein terminal residues and employ a {\textquotedblleft}tag-specific{\textquotedblright} 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 0.11 mg ml-1 and 0.48 mg ml-1 for 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 106 M-1 affinity constants and Qmax values of 19.11 {\textpm} 2.60 ug g-1 and 79.39 ug g-1 for 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.</p>
},
	keywords = {Affinity chromatography, Affinity ligand, Affinity tag, Combinatorial Chemistry, Green fluorescen t protein, Recombinant Proteins, Ugi reaction},
	issn = {00219673},
	doi = {10.1016/j.chroma.2016.10.017},
	url = {http://linkinghub.elsevier.com/retrieve/pii/S0021967316313498},
	author = {Pina, Ana Sofia and Carvalho, Sara and Dias, Ana Margarida G.C. and Guilherme, M{\'a}rcia and Pereira, Alice S. and Cara{\c c}a, Luciana T. and Ana Sofia Coroadinha and Lowe, Christopher R. and Roque, Ana Cec{\'\i}lia A}
}
