Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF

J. Enrique Cantu-Bustos, Teresa Vargas-Cortez, Jose Ruben Morones-Ramirez, Isaias Balderas-Renteria, David W. Galbraith, Megan M. McEvoy, Xristo Zarate

Research output: Research - peer-reviewArticle

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Abstract

Production of recombinant proteins in Escherichia coli has been improved considerably through the use of fusion proteins, because they increase protein solubility and facilitate purification via affinity chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified. GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein production in vivo showed that CusF produces large amounts of soluble protein with low levels of inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins can be purified with readily available IMAC resins charged with this metal ion, producing pure proteins after purification and tag removal. We therefore recommend the use of CusF as a viable alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli.

LanguageEnglish (US)
Pages61-65
Number of pages5
JournalProtein Expression and Purification
Volume121
DOIs
StatePublished - May 1 2016

Fingerprint

Recombinant Proteins
Carrier Proteins
Metals
Escherichia coli
Proteins
Green Fluorescent Proteins
Fluorescence
Ions
Periplasm
Protein Array Analysis
Inclusion Bodies
Protein Sorting Signals
Affinity Chromatography
Solubility
Copper
imidazoleacetic acid

Keywords

  • Affinity tag
  • CusF
  • Escherichia coli
  • Fusion protein

ASJC Scopus subject areas

  • Biotechnology

Cite this

Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF. / Cantu-Bustos, J. Enrique; Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Galbraith, David W.; McEvoy, Megan M.; Zarate, Xristo.

In: Protein Expression and Purification, Vol. 121, 01.05.2016, p. 61-65.

Research output: Research - peer-reviewArticle

Cantu-Bustos, J. Enrique ; Vargas-Cortez, Teresa ; Morones-Ramirez, Jose Ruben ; Balderas-Renteria, Isaias ; Galbraith, David W. ; McEvoy, Megan M. ; Zarate, Xristo. / Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF. In: Protein Expression and Purification. 2016 ; Vol. 121. pp. 61-65
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