Rapid isolation of nuclear transport-competent Xenopus nucleoplasmin produced in Escherichia coli strain BL21(DE3).
Nucleoplasmin is a thermostable karyophilic protein widely used in nuclear transport studies. An expression vector was constructed that contains a string of 10 histidine residues ligated, in frame, to the amino terminal end of the Xenopus nucleoplasmin gene. The vector was then transformed into Escherichia coli strain BL21(DE3). This strain possesses the gene for T7 RNA polymerase under control of the lacUV5 promoter. The induction of the RNA polymerase and subsequent production of nucleoplasmin occurs after exposure to isopropyl-beta-D-thiogalactopyranoside. The nucleoplasmin, produced in milligram quantities per liter of culture, is then isolated by a rapid purification method that includes metal chelation chromatography to purify the oligohistidine-linked nucleoplasmin. Nuclear transport studies indicate that fluorescently labeled nucleoplasmin is translocated to the nuclear interior of permeabilized V79A03 cells, while nucleoplasmin that lacks a nuclear localization signal (core nucleoplasmin) is not imported. The use of this method to produce nuclear transport-competent nucleoplasmin avoids the lengthy purification procedure used to isolate nucleoplasmin from Xenopus laevis oocytes as well as the cost of purchasing and maintaining a toad colony.
Kalinich
J F
JF
Radiation Biochemistry Department, Armed Forces Radiobiology Research Institute, Bethesda, Maryland 20889-5603.
McClain
D E
DE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D056489:Nucleoplasmins; D010750:Phosphoproteins; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014170:Transformation, Genetic; D014982:Xenopus laevis
D009687:Nuclear Proteins; D056489:Nucleoplasmins; D010750:Phosphoproteins; D011994:Recombinant Proteins