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

2 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

Fingerprint

Recombinant Proteins
Tryptophan
Purification
Ligands
Proteins
Fusion reactions
Fluorescence
Inclusion Bodies
Electric fuses

Keywords

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

Cite this

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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 0.11 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.",
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Tryptophan tags and de novo designed complementary affinity ligands for the expression and purification of recombinant proteins. / Pina, Ana Sofia; Carvalho, Sara ; Dias, Ana Margarida G C; Guilherme, Márcia; Pereira, Maria Alice Santos; Caraça, Luciana T.; Coroadinha, Ana Sofia; Lowe, Christopher R; Roque, A. Cecília A.

In: Journal Of Chromatography A, Vol. 1472, 11.11.2016, p. 55-65.

Research output: Contribution to journalArticle

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AU - Pina, Ana Sofia

AU - Carvalho, Sara

AU - Dias, Ana Margarida G C

AU - Guilherme, Márcia

AU - Pereira, Maria Alice Santos

AU - Caraça, Luciana T.

AU - Coroadinha, Ana Sofia

AU - Lowe, Christopher R

AU - Roque, A. Cecília A

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