Cloning, expression, purification and crystallization of NHR3 domain from acute myelogenous leukemia-related protein AML1-ETO.
The t(8;21) translocation is one of the most frequent chromosome abnormalities in acute myeloid leukemia. This translocation creates a fusion between the acute myelogenous leukemia 1 (AML1, a transcription factor) gene on chromosome 21 and the eight-twenty-one (ETO, a zinc finger nuclear protein) gene on chromosome 8, leading to the repression of certain AML1 target genes. We cloned NHR3 domain coding fragment into vector pGEX-6p-1 using PCR and obtained recombinant plasmid pGEX-6p-1-NHR3, which can be induced to stably overexpress fusion protein in E. coli. Through the purification on GST affinity chromatography column and PreScission protease cleavage, a large amount of NHR3 protein with high purity was obtained. In order to avoid possible interference of some strong negative charged molecules, NHR3 protein was further purified by Mono Q anion exchange chromatography. The NHR3 crystals were grown with hanging drop/vapor diffusion method and the first crystals appeared after four weeks at 18 degrees in 0.2 M Tris-sodium citrate dihydrate, 0.1 M sodium cacodylate, pH 6.5, and 30% iso-propanol (V/V). ESI mass spectrum showed that the molecular weight of this domain was in agreement with its primary structure sequence prediction, and circular dichroism spectral data (190-250 nm) showed that NHR3 had a well-defined secondary structure of 25.9% alpha-helix, 23.2% random coil and 50.9% turn, which was consistent with GOV4 software prediction.
Yang
Hai-Tao
HT
Shanghai Institute of Hematology, State Key Laboratory of Medical Genomics, Shanghai Second Medical University, Shanghai 200023, China. sjchen@stn.sh.cn
Wu
Dong-Hui
DH
Xue
Xiao-Yu
XY
Liang
Wen-Xue
WX
Miao
Xiao-Yu
XY
Pang
Hai
H
Chen
Sai-Juan
SJ
D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D050676:Core Binding Factor Alpha 2 Subunit; D003460:Crystallization; D004273:DNA, Neoplasm / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000235:genetics; D015514:Oncogene Proteins, Fusion / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D017434:Protein Structure, Tertiary; D000075142:RUNX1 Translocation Partner 1 Protein; D021241:Spectrometry, Mass, Electrospray Ionization; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*
C107844:AML1-ETO fusion protein, human; D050676:Core Binding Factor Alpha 2 Subunit; D004273:DNA, Neoplasm; D015514:Oncogene Proteins, Fusion; D000075142:RUNX1 Translocation Partner 1 Protein; D014157:Transcription Factors