title: Biotin-ubiquitin tagging of mammalian proteins in Escherichia coli.
abstract: Ubiquitin has been used in protein expression for enhancing yields and biological activities of recombinant proteins. Biotin binds tightly and specifically to avidin and has been widely utilized as a tag for protein purification and monitoring. Here, we report a versatile system that takes the advantages of both biotin and ubiquitin for protein expression, purification, and monitoring. The tripartite system contained coding sequences for a leader biotinylation peptide, ubiquitin, and biotin holoenzyme synthetase in two reading frames under the control of T7 promoter. The expression and purification of several large mammalian enzymes as biotin-ubiquitin fusions were accomplished including human ubiquitin activating enzyme, SUMO activating enzymes, and aspartyl-tRNA synthetase. Expressed proteins were purified by one-step affinity column chromatography on monomeric avidin columns and purified proteins exhibited active function. Additionally, the ubiquitin protein hydrolase UBP41, expressed and purified as biotin-UBP41, efficiently and specifically cleaved off the biotin-ubiquitin tag from biotin-ubiquitin fusions to produce unmodified proteins. The present expression system should be useful for the expression, purification, and functional characterization of mammalian proteins and the construction of protein microarrays.
authors.affiliation: Department of Chemistry, Georgetown University, Washington, DC 20057, USA.
authors.forename: Tao
authors.initials: T
authors.lastname: Wang
authors.forename: Evgenij
authors.initials: E
authors.lastname: Evdokimov
authors.forename: Kwabena
authors.initials: K
authors.lastname: Yiadom
authors.forename: Zhengyin
authors.initials: Z
authors.lastname: Yan
authors.forename: P Boon
authors.initials: PB
authors.lastname: Chock
authors.forename: David C H
authors.initials: DC
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001226:Aspartate-tRNA Ligase / Q000235:genetics / Q000378:metabolism; D001710:Biotin / Q000235:genetics / Q000378:metabolism*; D019921:Biotinylation; D004926:Escherichia coli / Q000235:genetics; D020690:GTPase-Activating Proteins / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D044764:Ubiquitin-Activating Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D020690:GTPase-Activating Proteins; C092317:RANGAP1 protein, human; D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; D025801:Ubiquitin; D001710:Biotin; D014357:Trypsin; D001226:Aspartate-tRNA Ligase; D044764:Ubiquitin-Activating Enzymes