Tryptophan tags and de novo designed complementary affinity ligands for the expression and purification of recombinant proteins

Ana Sofia Pina, Sara Carvalho, Ana Margarida G C Dias, Márcia Guilherme, Maria Alice Santos Pereira, Luciana T. Caraça, Ana Sofia Coroadinha, Christopher R Lowe, A. Cecília A Roque

Research output: Contribution to journalArticle

4 Citations (Scopus)

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 0.11 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 ± 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.

Original languageEnglish
Pages (from-to)55-65
Number of pages11
JournalJournal Of Chromatography A
Volume1472
DOIs
Publication statusPublished - 11 Nov 2016

Keywords

  • Affinity chromatography
  • Affinity ligand
  • Affinity tag
  • Combinatorial chemistry
  • Green fluorescent protein
  • Recombinant proteins
  • Ugi reaction

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