Biotin-ubiquitin tagging of mammalian proteins in Escherichia coli.
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.
Wang
Tao
T
Department of Chemistry, Georgetown University, Washington, DC 20057, USA.
Evdokimov
Evgenij
E
Yiadom
Kwabena
K
Yan
Zhengyin
Z
Chock
P Boon
PB
Yang
David C H
DC
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
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