High-yield expression in Escherichia coli and purification of mouse ubiquitin-activating enzyme E1.
Research in the ubiquitin field requires large amounts of ubiquitin-activating enzyme (E1) for in vitro ubiquitination assays. Typically, the mammalian enzyme is either isolated from natural sources or produced recombinantly using baculovirus/insect cell protein expression systems. Escherichia coli is seldom used to produce mammalian E1 probably due to the instability and insolubility of this high-molecular mass protein. In this report, we show that 5-10 mg of histidine-tagged mouse E1 can be easily obtained from a 1 l E. coli culture. A low temperature during the protein induction step was found to be critical to obtain an active enzyme.
Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.
Andreia F
AF
Carvalho
Manuel P
MP
Pinto
Cláudia P
CP
Grou
Rui
R
Vitorino
Pedro
P
Domingues
Fumiaki
F
Yamao
Clara
C
Sá-Miranda
Jorge E
JE
Azevedo
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D057075:Enzyme Assays; D004926:Escherichia coli / Q000737:chemistry* / Q000201:enzymology* / Q000235:genetics; D006639:Histidine / Q000031:analogs & derivatives / Q000737:chemistry / Q000235:genetics; D008099:Liver / Q000737:chemistry / Q000166:cytology; D051379:Mice; D000074435:Peroxisome-Targeting Signal 1 Receptor; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D044764:Ubiquitin-Activating Enzymes / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*
D000074435:Peroxisome-Targeting Signal 1 Receptor; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D006639:Histidine; D044764:Ubiquitin-Activating Enzymes