TY - JOUR
T1 - Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF
AU - Cantu-Bustos, J. Enrique
AU - Vargas-Cortez, Teresa
AU - Morones-Ramirez, Jose Ruben
AU - Balderas-Renteria, Isaias
AU - Galbraith, David W.
AU - McEvoy, Megan M.
AU - Zarate, Xristo
N1 - Funding Information:
We thank Mexico's Consejo Nacional de Ciencia y Tecnologia (CONACYT) for financial support provided to JECB and TVC during their graduate studies. We thank undergraduate students Kevin D. Cano del Villar, Huberto Hernandez Davila, Jesus A. del Moral Salgado, and Oscar F. Ramirez Mata for their help with DNA plasmid construction and protein purification experiments. This work was funded by CONACYT Grant CB 2012-179774-B awarded to XZ, and NIH Grant R01 079192 to MMM.
Publisher Copyright:
© 2016 Elsevier Inc. All rights reserved.
PY - 2016/5/1
Y1 - 2016/5/1
N2 - 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.
AB - 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.
KW - Affinity tag
KW - CusF
KW - Escherichia coli
KW - Fusion protein
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U2 - 10.1016/j.pep.2016.01.007
DO - 10.1016/j.pep.2016.01.007
M3 - Article
C2 - 26805756
AN - SCOPUS:84955501924
SN - 1046-5928
VL - 121
SP - 61
EP - 65
JO - Protein Expression and Purification
JF - Protein Expression and Purification
ER -