RESUMEN
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.
Asunto(s)
Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/aislamiento & purificación , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/aislamiento & purificación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas de Transporte de Catión/química , Proteínas Transportadoras de Cobre , Escherichia coli/genética , Proteínas de Escherichia coli/química , Fluorescencia , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Histidina/química , Metales/química , Proteínas Recombinantes de Fusión/genética , SolubilidadRESUMEN
Fusion proteins have become essential for the expression and purification of recombinant proteins in Escherichia coli. The metal-binding protein CusF has shown several features that make it an attractive fusion protein and affinity tag: "Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF" (Cantu-Bustos et al., 2016 [1]). Here we present accompanying data from protein expression experiments; we tested different protein tags, temperatures, expression times, cellular compartments, and concentrations of inducer in order to obtain soluble protein and low formation of inclusion bodies. Additionally, we present data from the purification of the green fluorescent protein (GFP) tagged with CusF, using Ag(I) metal affinity chromatography.