title: Production of recombinant proteins in Escherichia coli tagged with the fusion protein CusF3H.
abstract: Recombinant protein expression in the bacterium Escherichia coli still is the number one choice for large-scale protein production. Nevertheless, many complications can arise using this microorganism, such as low yields, the formation of inclusion bodies, and the requirement for difficult purification steps. Most of these problems can be solved with the use of fusion proteins. Here, the use of the metal-binding protein CusF3H+ is described as a new fusion protein for recombinant protein expression and purification in E. coli. We have previously shown that CusF produces large amounts of soluble protein, with low levels of formation of inclusion bodies, and that proteins can be purified using IMAC resins charged with Cu(II) ions. CusF3H+ is an enhanced variant of CusF, formed by the addition of three histidine residues at the N-terminus. These residues then can bind Ni(II) ions allowing improved purity after affinity chromatography. Expression and purification of Green Fluorescent Protein tagged with CusF3H+ showed that the mutation did not alter the capacity of the fusion protein to increase protein expression, and purity improved considerably after affinity chromatography with immobilized nickel ions; high yields are obtained after tag-removal since CusF3H+ is a small protein of just 10 kDa. Furthermore, the results of experiments involving expression of tagged proteins having medium to large molecular weights indicate that the presence of the CusF3H+ tag improves protein solubility, as compared to a His-tag. We therefore endorse CusF3H+ as a useful alternative fusion protein/affinity tag for production of recombinant proteins in E. coli.
authors.affiliation: Universidad Autonoma de Nuevo Leon, Facultad de Ciencias Quimicas, San Nicolas de los Garza, NL 66455, 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 66455, Mexico; Universidad Autonoma de Nuevo Leon, Centro de Investigacion en Biotecnologia y Nanotoxicologia, Facultad de Ciencias Quimicas, Parque de Investigacion e Innovacion Tecnologica, Apodaca, NL 66629, 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 66455, Mexico; Universidad Autonoma de Nuevo Leon, Centro de Investigacion en Biotecnologia y Nanotoxicologia, Facultad de Ciencias Quimicas, Parque de Investigacion e Innovacion Tecnologica, Apodaca, NL 66629, Mexico.
authors.forename: Isaias
authors.initials: I
authors.lastname: Balderas-Renteria
authors.affiliation: Universidad Autonoma de Nuevo Leon, Facultad de Ciencias Quimicas, San Nicolas de los Garza, NL 66455, Mexico; Universidad Autonoma de Nuevo Leon, Centro de Investigacion en Biotecnologia y Nanotoxicologia, Facultad de Ciencias Quimicas, Parque de Investigacion e Innovacion Tecnologica, Apodaca, NL 66629, Mexico. Electronic address: xristo.zaratekl@uanl.edu.mx.
authors.forename: Xristo
authors.initials: X
authors.lastname: Zarate
mesh_terms: D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D051792:Basic Helix-Loop-Helix Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D027682:Cation Transport Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D000079922:Copper Transport Proteins; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D046939:Synechocystis / Q000235:genetics* / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D001426:Bacterial Proteins; D051792:Basic Helix-Loop-Helix Proteins; D027682:Cation Transport Proteins; D000079922:Copper Transport Proteins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; C506213:CusF protein, E coli; C116162:PIF3 protein, Arabidopsis
keywords: Affinity tag; CusF3H+; Escherichia coli; Fusion protein; Recombinant proteins