Purification of human transcription factors Nanog and Sox2, each in complex with Skp, an Escherichia coli periplasmic chaperone.
Nanog and Sox2 are key transcriptional factors involved in self-renewal and pluripotency of stem cells in human and other mammals. Nanog and Sox2 contain homeodomain (HD) and high-mobility group (HMG) DNA-binding domain, respectively, for targeting them to their regulatory regions and the other regions with transactivation function by providing sites for recruiting other transcriptional regulators. To gain insights in the biochemical and biophysical characteristics of the other regions of Nanog and Sox2, we have tried to overproduce and purify full length wild-type human Nanog and Sox2 expressed in Escherichia coli. Interestingly, we found that Nanog and Sox2 were individually stabilized by tight interaction with Skp, an E. coli periplasmic chaperone, thereby enabling stable over-expression and purification of Nanog and Sox2, each in complex with Skp. Purified Skp complexes of Nanog and Sox maintained DNA-binding activity toward its cognate DNA sequence. A similar approach may be applicable for some other mammalian proteins that are unstable or difficult to over-express in E. coli.
Ha
Sung Chul
SC
Department of Chemistry, University of California, Berkeley, CA 94720, USA.
Pereira
Jose Henrique
JH
Jeong
Jin Hee
JH
Huh
Jin Hoe
JH
Kim
Sung-Hou
SH
D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D018832:Molecular Chaperones / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000302:isolation & purification* / Q000378:metabolism; D000071317:Nanog Homeobox Protein; D011485:Protein Binding; D055550:Protein Stability; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D018398:Homeodomain Proteins; D018832:Molecular Chaperones; D046912:Multiprotein Complexes; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D011994:Recombinant Proteins; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors; C072721:Skp protein, E coli