title: Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF.
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.
authors.affiliation: Universidad Autonoma de Nuevo Leon, Facultad de Ciencias Quimicas, San Nicolas de los Garza, NL 66451, Mexico.
authors.forename: J Enrique
authors.initials: JE
authors.lastname: Cantu-Bustos
authors.affiliation: Universidad Autonoma de Nuevo Leon, Facultad de Ciencias Quimicas, San Nicolas de los Garza, NL 66451, Mexico.
authors.forename: Teresa
authors.initials: T
authors.lastname: Vargas-Cortez
authors.affiliation: Universidad Autonoma de Nuevo Leon, Facultad de Ciencias Quimicas, San Nicolas de los Garza, NL 66451, Mexico.
authors.forename: Jose Ruben
authors.initials: JR
authors.lastname: Morones-Ramirez
authors.affiliation: Universidad Autonoma de Nuevo Leon, Facultad de Ciencias Quimicas, San Nicolas de los Garza, NL 66451, Mexico.
authors.forename: Isaias
authors.initials: I
authors.lastname: Balderas-Renteria
authors.affiliation: BIO5 Institute and School of Plant Sciences, University of Arizona, Tucson, AZ 85721, USA.
authors.forename: David W
authors.initials: DW
authors.lastname: Galbraith
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721, USA.
authors.forename: Megan M
authors.initials: MM
authors.lastname: McEvoy
authors.affiliation: Universidad Autonoma de Nuevo Leon, Facultad de Ciencias Quimicas, San Nicolas de los Garza, NL 66451, Mexico. Electronic address: xristo.zaratekl@uanl.edu.mx.
authors.forename: Xristo
authors.initials: X
authors.lastname: Zarate
mesh_terms: D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D000079922:Copper Transport Proteins; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D008670:Metals / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D012995:Solubility
chemical_list: D002352:Carrier Proteins; D027682:Cation Transport Proteins; D000079922:Copper Transport Proteins; C506213:CusF protein, E coli; D029968:Escherichia coli Proteins; D008670:Metals; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D006639:Histidine
keywords: Affinity tag; CusF; Escherichia coli; Fusion protein