title: Production of a soluble and functional recombinant streptavidin in Escherichia coli.
abstract: The cDNA for streptavidin (residues 15-159) was subcloned into an expression vector in fusion at the N-terminus with the T7-tag (12 residues). Conditions were found to express the protein in Escherichia coli in a soluble, assembled, and active form. The protein was purified in two simple steps which involved heating at 75 degreesC and affinity chromatography on iminobiotin agarose. The purified protein was obtained in yields of 70 mg per liter of bacterial culture. Electron spray mass spectrometry analysis showed that the recombinant streptavidin had the expected molecular mass without covalent modifications. ELISA and surface plasmon resonance analyses showed it to be functionally analogous to the natural streptavidin. This appears to be an improvement over the reported methods of recombinant streptavidin production which involve protein renaturation or the use of eukaryotic expression systems.
authors.affiliation: Dibit, Department of Biological and Technological Research, San Raffaele Scientific Institute, Milan, 20132, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Gallizia
authors.forename: C
authors.initials: C
authors.lastname: de Lalla
authors.forename: E
authors.initials: E
authors.lastname: Nardone
authors.forename: P
authors.initials: P
authors.lastname: Santambrogio
authors.forename: A
authors.initials: A
authors.lastname: Brandazza
authors.forename: A
authors.initials: A
authors.lastname: Sidoli
authors.forename: P
authors.initials: P
authors.lastname: Arosio
mesh_terms: D001710:Biotin / Q000378:metabolism; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D005656:Fungal Proteins / Q000302:isolation & purification; D015870:Gene Expression / Q000235:genetics; D013058:Mass Spectrometry; D009842:Oligopeptides / Q000235:genetics; D011485:Protein Binding / Q000502:physiology; D011994:Recombinant Proteins / Q000302:isolation & purification*; D019809:Streptavidin / Q000737:chemistry*; D013302:Streptomyces / Q000737:chemistry*
chemical_list: D005656:Fungal Proteins; D009842:Oligopeptides; D011994:Recombinant Proteins; C037841:tyrosyl-alanyl-glycine; D001710:Biotin; D019809:Streptavidin