Tryptophan tags and de novo designed complementary affinity ligands for the expression and purification of recombinant proteins
| Autor: | Sara Micaela Pereira Carvalho, Márcia Guilherme, A. Cecília A. Roque, Alice S. Pereira, Ana M. G. C. Dias, Ana S. Coroadinha, Christopher R. Lowe, Ana Sofia Pina, Luciana T. Caraça |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Protein Denaturation Recombinant Fusion Proteins Green Fluorescent Proteins Ligands 01 natural sciences Biochemistry Chromatography Affinity Protein Refolding Analytical Chemistry Green fluorescent protein 03 medical and health sciences FLAG-tag Affinity chromatography Amino Acid Sequence Tandem affinity purification Inclusion Bodies Chromatography Chemistry 010401 analytical chemistry Organic Chemistry Tryptophan General Medicine Isotope-coded affinity tag Fusion protein 0104 chemical sciences 030104 developmental biology Solubility Affinity electrophoresis Myc-tag |
| Zdroj: | Journal of chromatography. A. 1472 |
| ISSN: | 1873-3778 |
| Popis: | 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-1 and 0.48mgml-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 106M-1 affinity constants and Qmax values of 19.11±2.60ugg-1 and 79.39ugg-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. |
| Databáze: | OpenAIRE |
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