title: Enhanced expression, detection and purification of recombinant proteins using RNA stem loop and tandem fusion tags.
abstract: The creation of a double His-tag fusion that forms a RNA stem loop in the mRNA encoding the N-terminus of the target protein is a novel approach for the enhancement of expression, purification, and detection of a recombinant protein. Compared to a single His-tag fusion, a tandem His-tag fusion RNA stem loop, located downstream of the constitutive groE and Ch promoters, enhanced heterologous gene expression in Brucella, Salmonella, and Escherichia. We demonstrated one-step detection and purification of recombinant green fluorescence protein (GFP) directly from Brucella spp. without using Escherichia coli as an expression host. The amount of purified GFP using the tandem His-tag RNA stem loop increased more than threefold; moreover, the sensitivity of detection increased more than fourfold in comparison to the single His-tag fusion form. This method has the potential to significantly improve heterologous gene expression and high-throughput protein synthesis and purification.
authors.affiliation: Department of Biomedical Sciences and Pathobiology, Center of Molecular Medicine and Infectious Diseases, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic and State University, 1410 Prices Fork Rd, Blacksburg, VA 24060, USA.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Seleem
authors.forename: Mohammed
authors.initials: M
authors.lastname: Ali
authors.forename: M W Abd
authors.initials: MW
authors.lastname: Al-Azeem
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Boyle
authors.forename: Nammalwar
authors.initials: N
authors.lastname: Sriranganathan
mesh_terms: D002002:Brucella / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine; D012313:RNA; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D012475:Salmonella / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D006639:Histidine; D012313:RNA