title: Use of rifampicin in T7 RNA polymerase-driven expression of a plant enzyme: rifampicin improves yield and assembly.
abstract: Expression systems based on high selectivity and activity of T7 RNA polymerase and presence of a strong T7 promoter have been commonly used for cloning and expression of various recombinant proteins in Escherichia coli. When the expression system is designed in such a way that the produced protein is not being transferred into periplasm, bacterial cells must be lysed in order to isolate and purify the protein. The final yield and quality of the synthesized protein then depend on various factors, protein size, amino acid sequence, solubility in cytoplasm, and folding requirements among them. The yield in the T7 RNA polymerase/promoter system can be positively influenced by use of rifampicin. In this report we demonstrate usefulness of the antibiotic in detail. We describe rifampicin-enhanced expression of a plant cytokinin-specific beta-glucosidase. Two bacterial cultures are compared, one expressing the enzyme without and one in the presence of rifampicin. The antibiotic not only increased the yield of the recombinant protein, which seems to be a general phenomenon, but also favored the final assembly of the protein's subunits into a catalytically active dimer form.
authors.affiliation: Faculty of Science, Masaryk University, Kotlárská 2, Brno, 611 37, Czech Republic.
authors.forename: A
authors.initials: A
authors.lastname: Kuderová
authors.forename: E
authors.initials: E
authors.lastname: Nanak
authors.forename: M
authors.initials: M
authors.lastname: Truksa
authors.forename: B
authors.initials: B
authors.lastname: Brzobohatý
mesh_terms: D015153:Blotting, Western; D003583:Cytokinins / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012293:Rifampin / Q000494:pharmacology*; D014764:Viral Proteins; D001617:beta-Glucosidase / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*
chemical_list: D003583:Cytokinins; D011994:Recombinant Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; C071257:cytokinin-beta-glucosidase; D001617:beta-Glucosidase; D012293:Rifampin