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重组信号调节蛋白α(SIRPα)胞外段及其高亲和力突变体的表达、纯化与活性鉴定 被引量：2

Expression,purification and characterization of human SIRPα extracellular domain and its high affinity mutant
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摘要 制备野生型信号调节蛋白α(SIRPα)胞外段和高亲和力突变体,并进行纯化条件优化与结合活性鉴定。通过全基因合成获得SIRPα-wt和高亲和力突变体SIRPα-cvl的全基因;利用分子克隆技术将其连入表达载体,最终通过大肠杆菌BL21(DE3)工程菌株表达Trx-SIRPα-wt和Trx-SIRPα-cvl两种融合蛋白。通过肠激酶酶切去除融合标签以及多次层析纯化后,利用质谱技术分析目的蛋白所在组分。经SDS-PAGE电泳显示,最终获得了纯度高于90%的SIRPα-wt和SIRPα-cvl,与CD47结合活性结果表明两种蛋白都能与CD47-Fc融合蛋白结合。据此,本研究成功表达并纯化得到了具有配体结合活性的SIRPα-wt和其高亲和力突变体SIRPα-cvl两种重组蛋白。 With the known protein sequence,we synthesize the DNA of SIRPα-wt and its high affinity mutant SIRPα-cvl.And then we expressed the fusion proteins Trx-SIRPα-wt and Trx-SIRPα-cvl by E.coli BL21（DE3）.After the fusion partner was removed by enteropeptidase,SIRPα-wt and SIRPα-cvl were purified by Ni-Affinity Chromatography and Q-Ion-Exchange Chromatography.According to SDS-PAGE analysis,the purity of both SIRPα were higher than 90%,and the ELISA analysis showed that both of them could bind with fusion protein CD47-Fc.The molecular weight of the purified SIRPα-wt and SIRPα-cvl were also confirmed by LC-MS.Taken together,we have successfully expressed and purified the recombinant protein SIRPα-wt and SIRPα-cvl without extra residues.
作者 徐进 刘涛 赵自叶 彭晓芸 夏懋 郭怀祖 张大鹏 钱卫珠 王皓
机构地区 上海交通大学药学院 第二军医大学肿瘤研究所 聊城大学药学院 华南理工大学生物科学与工程学院
出处 《现代免疫学》 CAS CSCD 北大核心 2014年第5期353-357,共5页 Current Immunology
基金 863计划项目(2012AA02A304) 上海市细胞工程重点实验室项目
关键词 SIRPα CD47 融合蛋白 高亲和力突变体 蛋白纯化 SIRPα CD47 fusion protein high affinity mutant protein purification
分类号 R392 [医药卫生—免疫学]
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参考文献16

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同被引文献22

  • 1van den Berg TK, van Beek EM, Buhring HJ, et al. A no-menclature for signal regulatory protein family members[J].J Immunol, 2005,175 : 7788-7789.
  • 2Barclay AN, van den Berg TK. The interaction between sig-nal regulatory protein alpha (SIRPa) and CD47 : structure,function, and therapeutic target [J]. Ann Rev Immunol.2014, 32: 25-50.
  • 3Adams S, van der Laan LJ, Vernon-Wilson E, et al. Signal-regulatory protein is selectively expressed by myeloid andneuronal cells[J]. J Immunol, 1998,161 : 1853-1859.
  • 4Veillette A,Thibaudeau E,Latour S. High expression of in-hibitory receptor SHPS-1 and its association with protein-tyrosine phosphatase SHP-1 in macrophages [ J ]. J BiolChem, 1998, 273: 22719-22728.
  • 5Seiffert M,Cant C,Chen Z,et al. Human signal-regulatoryprotein is expressed on normal, but not on subsets of leuke-mic myeloid cells and mediates cellular adhesion involving itscounterreceptor CD47[J]. Blood, 1999,94: 3633-3643.
  • 6Seiffert M,Brossart P, Cant C, et al. Signal-regulatory pro-tein alpha (SIRPalpha) but not SIRPbeta is involved in T-cellactivation, binds to CD47 with high affinity, and is expressedon immature CD34 ( + ) CD38 ( - ) hematopoietic cells[J],Blood, 2001, 97: 2741-2749.
  • 7Latour S,Tanaka H,Demeure C,et al. Bidirectional nega-tive regulation of human T and dendritic cells by CD47 and itscognate receptor signal-regulator protein-alpha: down-regula-tion of IL-12 responsiveness and inhibition of dendritic cell ac-tivation[J], J Immunol, 2001,167 : 2547-2554.
  • 8Jaiswal S,Jamieson CH, Pang WW, et al. CD47 is upregu-lated on circulating hematopoietic stem cells and leukemiacells to avoid phagocytosis[J]. Cell,2009,138 : 271-285.
  • 9Chao MP, Alizadeh AA,Tang C,et al, Anti-CD47 antibodysynergizes with rituximab to promote phagocytosis and eradi-cate non-Hodgkin lymphoma[J]. Cell, 2010,142 : 699-713.
  • 10Sick E, Jeanne A, Schneider C, etal. CD47 update; a multi-faceted actor in the tumour microenvironment of potentialtherapeutic interest[J]. Brit J Pharmacol, 2012, 167 : 1415-1430.

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现代免疫学
2014年 第5期

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