Please wait a minute...

中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2011, Vol. 31 Issue (12): 1-9    DOI:
    
Eucaryotic Expression and Purification of DEK Protein and Its Activity Detection
GUO Hai-hong, YANG Xin, GUO Fang, HU Hong-gang
College of Life Sciences and Bioengineering, School of Science, Beijing Jiaotong University, Beijing 100044, China
Download: HTML   PDF(731KB) HTML
Export: BibTeX | EndNote (RIS)      

Abstract  

It was chosen the Bac-To-Bac® Baculovirus Expression System to express DEK protein, and the protein was purified under native condition. First, the full-length DEK gene was amplified from pMD18-T-DEK, and then inserted into plasmid pFastBacI of the Bac-To-Bac® Baculovirus expression system to form plasmid pFastBacI-DEK. Recombinant shuttle vector named Bacmid-DEK was obtained in E.coli DH10Bac through transformation. After transfection with Cellfectin® II Reagent, recombinant Baculovirus (AcNPV-DEK) was developed in Sf9 cells. Then this viral stock was used to infect new Sf9 cells to generate a high-titer baculoviral stock. After that, the baculoviral stock can be used to infect Sf9 cells for DEK expression. The DEK protein was purified under native conditions using Ni-NTA agarose. The specific 50 kDa band was showed by SDS-PAGE and Western blotting, which indicated that DEK protein was expressed in Sf9 cells and the highly purified His-DEK proteins were obtained. Using the purified His-DEK, the Electrophoretic mobility shift assay (EMSA)was performed to test the binding activity of His-DEK to DNA molecules. The results showed that His-DEK and His-CDB (expressed by E.coli BL21 strain) could bind DNA molecules, especially, preferring the supercoiled form in vitro; meanwhile, the DNA binding activity of His-DEK was higher than His-CDB. It is reported that phosphorylation of DEK protein could inhibit the binding of DEK to DNA. The expressed His-DEK protein in Sf9 cells was phosphorylated, so the His-DEK protein was dephosphorylated using λ-PPase. The result of EMSA showed that the DNA binding activity of dephosphorylated His-DEK protein increased than the phosphorylated protein.

Key words： DEK protein      Baculovirus expression system      Electrophoretic mobility shift assay     
Received: 22 August 2011      Published: 25 December 2011
ZTFLH:  Q819  
Service
E-mail this article
Add to my bookshelf
Add to citation manager
E-mail Alert
RSS
Articles by authors
Cite this article:

GUO Hai-hong, YANG Xin, GUO Fang, HU Hong-gang. Eucaryotic Expression and Purification of DEK Protein and Its Activity Detection. China Biotechnology, 2011, 31(12): 1-9.

URL:

https://manu60.magtech.com.cn/biotech/     OR     https://manu60.magtech.com.cn/biotech/Y2011/V31/I12/1


[1] Hu H G, Scholten I, Gruss C, et al. The distribution of the DEK protein in mammalian chromatin. Biochem Biophys Res Commun, 2007, 358(4):1008-1014.

[2] Waldmann T, Baack M, Richter N, et al. Structure-specific binding of the proto-oncogene protein DEK to DNA. Nucleic Acids Res, 2003, 31(23):7003-7010.

[3] Waldmann T, Eckerich C, Baack M, et al. The ubiquitous chromatin protein DEK alters the structure of DNA by introducing positive supercoils. J Biol Chem, 2002, 277(28):24988-24994.

[4] Waldmann T, Scholten I, Kappes F, et al. The DEK protein-an abundant and ubiquitous constituent of mammalian chromatin. Gene, 2004, 343(1):1-9.

[5] von Lindern M, Fornerod M, van Baal S, et al. The translocation (6;9), associated with a specific subtype of acute myeloid leukemia, results in the fusion of two genes, dek and can, and the expression of a chimeric, leukemia-specific dek-can mRNA. Mol Cell Biol, 1992, 12(4):1687-1697.

[6] Soekarman D, von Lindern M, Daenen S, et al. The translocation (6;9) (p23;q34) shows consistent rearrangement of two genes and defines a myeloproliferative disorder with specific clinical features. Blood, 1992, 79(11):2990-2997.

[7] Dong X, Wang J, Kabir F N, et al. Autoantibodies to DEK oncoprotein in human inflammatory disease. Arthritis Rheum, 2000, 43(1):85-93.

[8] Sierakowska H, Williams K R, Szer I S, et al. The putative oncoprotein DEK, part of a chimera protein associated with acute myeloid leukaemia, is an autoantigen in juvenile rheumatoid arthritis. Clin Exp Immunol, 1993, 94(3):435-439.

[9] Dong X, Michelis M A, Wang J, et al. Autoantibodies to DEK oncoprotein in a patient with systemic lupus erythematosus and sarcoidosis. Arthritis Rheum, 1998, 41(8):1505-1510.

[10] Wichmann I, Respaldiza N, Garcia-Lozano J R, et al. Autoantibodies to DEK oncoprotein in systemic lupus erythematosus (SLE). Clin Exp Immunol, 2000, 119(3):530-532.

[11] Alexiadis V, Waldmann T, Andersen J, et al. The protein encoded by the proto-oncogene DEK changes the topology of chromatin and reduces the efficiency of DNA replication in a chromatin-specific manner. Genes Dev, 2000, 14(11):1308-1312.

[12] Kappes F, Fahrer J, Khodadoust M S, et al. DEK is a poly(ADP-ribose) acceptor in apoptosis and mediates resistance to genotoxic stress. Mol Cell Biol, 2008, 28(10):3245-3257.

[13] Campillos M, Garcia M A, Valdivieso F, et al. Transcriptional activation by AP-2alpha is modulated by the oncogene DEK. Nucleic Acids Res, 2003, 31(5):1571-1575.

[14] Faulkner N E, Hilfinger J M, Markovitz D M, Protein phosphatase 2A activates the HIV-2 promoter through enhancer elements that include the pets site. J Biol Chem, 2001, 276(28):25804-25812.

[15] Gamble M J, Fisher R P. SET PARP1 remove DEK from chromatin to permit access by the transcription machinery. Nat Struct Mol Biol, 2007, 14(6):548-555.

[16] Hollenbach A D, McPherson C J, Mientjes E J, et al. Daxx and histone deacetylase II associate with chromatin through an interaction with core histones and the chromatin-associated protein Dek. J Cell Sci, 2002, 115(Pt 16):3319-3330.

[17] Hu H G, Illges H, Gruss C, et al. Distribution of the chromatin protein DEK distinguishes active and inactive CD21/CR2 gene in pre-and mature B lymphocytes. Int Immunol, 2005, 17(6):789-796.

[18] Ko S I, Lee I S, Kim J Y, et al. Regulation of histone acetyltransferase activity of p300 and PCAF by proto-oncogene protein DEK. FEBS Lett, 2006, 580(13):3217-3222.

[19] Sammons M, Wan S S, Vogel N L, et al. Negative regulation of the RelA/p65 transactivation function by the product of the DEK proto-oncogene. J Biol Chem, 2006, 281(37):26802-26812.

[20] Le Hir H, Gatfield D, Izaurralde E, et al. The exon-exon junction complex provides a binding platform for factors involved in mRNA export and nonsense-mediated mRNA decay. Embo J, 2001, 20(17):4987-4997.

[21] McGarvey T, Rosonina E, McCracken S, et al. The acute myeloid leukemia-associated protein, DEK, forms a splicing-dependent interaction with exon-product complexes. J Cell Biol, 2000, 150(2):309-320.

[22] Soares L M, Zanier K, Mackereth C, et al. Intron removal requires proofreading of U2AF/3' splice site recognition by DEK. Science, 2006, 312(5782):1961-1965.

[23] Kim D W, Kim J Y, Choi S, et al. Transcriptional regulation of 1-cys peroxiredoxin by the proto-oncogen protein DEK. Mol Med Report, 2010, 3(5):877-881.

[24] Wise-Draper T M, Morreale R J, Morris T A, et al. DEK proto-oncogene expression interferes with the normal epithelial differentiation program. Am J Pathol, 2009, 174(1):71-81.

[25] Savli H, Aalto Y, Nagy B, et al. Gene expression analysis of 1,25(OH)2D3-dependent differentiation of HL-60 cells: a cDNA array study. Br J Haematol, 2002, 118(4):1065-1070.

[26] Ageberg M, Gullberg U, Lindmark A, The involvement of cellular proliferation status in the expression of the human proto-oncogene DEK. Haematologica, 2006, 91(2):268-269.

[27] Kim D W, Chae J I, Kim J Y, et al. Proteomic analysis of apoptosis related proteins regulated by proto-oncogene protein DEK. J Cell Biochem, 2009, 106(6):1048-1059.

[28] Lee K S, Kim D W, Kim J Y, et al. Caspase-dependent apoptosis induction by targeted expression of DEK in drosophila involves histone acetylation inhibition. J Cell Biochem, 2007, 103(4):1283-1293.

[29] Wise-Draper T M, Allen H V, Jones E E, et al. Apoptosis inhibition by the human DEK oncoprotein involves interference with p53 functions. Mol Cell Biol, 2006, 26(20):7506-7519.

[30] 华颖, 胡红刚, 彭向雷. DEK蛋白C末端DNA结合域的原核表达、纯化及其与DNA的结合活性. 中国生物工程杂志, 2009, 29(8):14-18. Hua Y, Hu H G, Pong X L. China Biotechnology, 2009, 29 (8): 14-18.
[1] FENG Pei-ping, ZHANG Lei, CHAI Xiu-li, ZHANG Hai-ling, ZHAO Jian-jun, HU Bo, BAI Xue, GAO Han, SHAO Xi-qun, YAN Xi-jun, ZHAO Quan, XU Lei. Expression of Nucleocapsid Protein of Canine Distemper Virus Base on Bac-to-Bac Baculovirus System[J]. China Biotechnology, 2012, 32(04): 60-66.
[2] ZHANG Mei, FU Yuan-hui, HE Jin-sheng, LU Yan-yan, ZHENG Xian-xian. Expression and Purification of Enhanced Green Fluorescent Protein Based on Baculovirus Expression System[J]. China Biotechnology, 2011, 31(5): 99-103.
[3] . Expression and Purification of Enhanced Green Fluorescent Protein Based on Baculovirus Expression System[J]. China Biotechnology, 2011, 31(05): 0-0.
[4] HUA Ying, HU Gong-Gang, BANG Xiang-Lei. Prokaryotic Expression,Purification and DNA Binding Activity of DEK Protein’s Carboxyterminal DNAbinding Region[J]. China Biotechnology, 2009, 29(08): 14-18.
主管：中国科学院 　主办：中国科学院文献情报中心 　中国生物技术发展中心 　中国生物工程学会