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ISSN 1672-9145 Acta Biochim Biophys Sin 2004, 36(8): 566–570 CN 31-1940/Q

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Cloning, Expression, Purification and Crystallization of NHR3 Domain from Acute Myelogenous Leukemia-related Protein AML1-ETO

Hai-Tao YANG#, Dong-Hui WU1#, Xiao-Yu XUE, Wen-Xue LIANG1, Xiao-Yu MIAO, Hai PANG*, and Sai-Juan CHEN1*

Laboratory of Structural Biology, Department of Biological Sciences and Biotechnology & Protein Sciences Laboratory of MOE, Tsinghua University, Beijing 100084, China; 1Shanghai Institute of Hematology, State Key Laboratory of Medical Genomics, Shanghai Second Medical University, Shanghai 200023, China

Abstract 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 ºC 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 (190250 nm) showed that NHR3 had a well-defined secondary structure of 25.9% a-helix, 23.2% random coil and 50.9% turn, which was consistent with GOV4 software prediction.

Key words AML1-ETO; NHR3; crystallization; ESI mass spectrum; circular dichroism spectrum; glutathione S-transferase (GST)

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Received: April 15, 2004 Accepted: June 10, 2004

#These authors contributed equally to this work

*Corresponding authors:

Sai-Juan CHEN: Tel, 86-21-64377859; Fax, 86-21-64743206; E-mail, [email protected]

Hai PANG: E-mail, [email protected]