Expression and Purification of Recombinant Human Histone #br# H3 and H4 for Cytotoxicity Analysis
ZHU Ming-Ang1), TAO Yue1), ZHANG Xiao-Qin1), CAO Qing2), MO Xi1)*
1) Key Laboratory of Pediatric Hematology & Oncology Ministry of Health, Pediatric Translational Medicine Institute,
2) Department of Infections, Shanghai Children’s Medical Center, Shanghai Jiaotong University
School of Medicine, Shanghai 200127, China
Histones normally exist in the cell nuclei, but are also released outside to the lumen forming so-called extracellular histones. Extracellular histones pose strong cytotoxicities to cells, particularly in the disease pathogenesis and progression of sepsis, acute lung injuries and rheumatoid arthritis. The quantification of extracellular histone levels in the patients’ plasma demands high quality histone standard. Otherwise, circulating histone levels will not be usable for the disease stage classification and evaluation of drug dosage. In laboratories, quantification of extracellular histones is used for exploring the mechanism of histone toxicity. In the present study, we expressed denatured histone H3 and H4 monomers in E. coli. After purification with affinity column, the refolded H3 and H4 monomers, as well as H3/H4 complex, could be obtained by either gradient dilution or dialysis. We found that the H3/H4 complex was the most stable form of recombinant histones. Treatment of HUVECs with 50 μg/mL recombinant H3/H4 complex induced about 80% cell death, equivalent to the treatment with 200 μg/mL calf thymus histones. Such cytotoxicity could be relieved dosedependently with human serum albumin from 0.625~10 mg/mL, which was similar to the situation with natural H3/H4 complex. Therefore, our recombinant H3/H4 complex was usable as a quantification standard for the study of histone-induced cytotoxicities.
朱明昂, 陶悦, 张小秦, 曹清, 莫茜. 重组人组蛋白H3和H4的表达纯化及其细胞毒性分析[J]. 中国生物化学与分子生物学报, 2016, 32(2): 191-198.
ZHU Ming-Ang, TAO Yue, ZHANG Xiao-Qin, CAO Qing, MO Xi. Expression and Purification of Recombinant Human Histone #br# H3 and H4 for Cytotoxicity Analysis. Chinese Journal of Biochemistry and Molecular Biol, 2016, 32(2): 191-198.
[1] Holdenrieder S, Stieber P. Clinical use of circulating nucleosomes[J]. Crit Rev Clin Lab Sci, 2009,46(1): 1-24
[2] Wildhagen KC, García de Frutos P, Reutelingsperger CP,et al. Nonanticoagulant heparin prevents histone-mediated cytotoxicity in vitro and improves survival in sepsis[J].Blood, 2014, 123 (7): 1098-1101
[3] Xu J, Zhang X, Pelayo R, et al.Extracellular histones are major mediators of death in sepsis[J]. Nat Med, 2009,15(11):1318-1321
[4] Hijiya N,Miyake K,Akashi S,et al.Possible involvement of toll-like receptor 4 in endothelial cell activation of larger vessels in response to lipopolysaccharide[J].Pathobiology,2002,70(1): 18-25
[5] Xu J, Zhang X, Monestier M,et al.Extracellular histones are mediators of death through TLR2 and TLR4 in mouse fatal liver injury[J]. J Immunol, 2011,187(5): 2626-2631
[6] Liu ZG, Ni SY, Chen GM, et al.Histones-mediated lymphocyte apoptosis during sepsis is dependent on p38 phosphorylation and mitochondrial permeability transition[J].PLoS One, 2013,8(10): e77131
[7] Wang F, Zhang N, Li B, et al. Heparin defends against the toxicity of circulating histones in sepsis[J]. Front Biosci (Landmark Ed), 2015,20: 1259-1270
[13] Pemberton AD,Brown JK,Inglis NF.Proteomic identification of interactions between histones and plasma proteins: implications for cytoprotection[J].Proteomics,2010,10(7): 1484-1493
[19]韩红芬,徐雪花,韩丽琴,等.脓毒症患儿血清白蛋白水平与预后的关系[J].海南医学(Han H F,Xu XH,Han LQ,et al. Relationship between serum albumin of children with sepsis and prognosis[J]. Hainan Med J), 2011, 22(9): 59-60