Expression, purification and biological characterization of the extracellular domain of CD40 from Pichia pastoris.
Expression, purification and biological characterization of the extracellular domain of CD40 from Pichia pastoris.
复制标题
毕赤酵母 CD40 胞外结构域的表达、纯化和生物学特性
DOI：
10.1186/s12896-016-0237-1
复制
发表时间：
2016-01-25
影响因子：
3.5
通讯作者：
Zhang X
中科院分区：
文献类型：
--
作者：
Zhan Y;Wei Y;Chen P;Zhang H;Liu D;Zhang J;Liu R;Chen R;Zhang J;Mo W;Zhang X
背景：CD 40又称Bp 50，是TNF受体超家族的新成员。基于其在多种生理和病理过程中的重要作用，CD 40信号通路已成为治疗移植、自身免疫性疾病和癌症的重要靶点。结果：通过密码子优化，获得了编码CD 40胞外区（CD 40-N）的DNA片段，并将其克隆到pPIC 9 K中，构建了毕赤酵母表达分泌菌株。SDS-PAGE和Western blotting分析表明，重组CD 40-N（27 kDa糖基化蛋白）可分泌到发酵液中。重组蛋白经Sephadex G-50分子排阻层析和Q Sepharose Fast Flow离子交换层析纯化，纯度达90%以上。最后，从3升上清液中获得120毫克纯度较高的蛋白质。结合试验（ITC 200试验）显示CD 40-N和CD 40激动剂抗体（G28-5）的直接相互作用。重组CD 40-N的生物活性通过其在体外破坏由CD 40激动剂抗体或CD 40配体激活的非经典NF-κB信号传导和抑制抗CD 40激动剂抗体诱导的BJAB细胞中TNF-α表达的能力来证实。此外，我们的数据表明，蛋白质具有治疗潜力，在治疗葡聚糖硫酸钠（DSS）诱导的结肠炎在vivo.Conclusions：结果表明，我们已经开发了使用巴斯德毕赤酵母的实验程序可用于生产大量的活性CD 40-N的研究和工业用途。我们获得的蛋白片段有可能用于研究甚至治疗炎症性疾病，如结肠炎。
Background:CD40, also called Bp50, is a novel member of the TNF receptor superfamily. Based on its important role in multiple physiological and pathological processes, the CD40 signaling pathway has become a vital target for treating transplantation, autoimmune diseases and cancers. This study generated a protein fragment that disrupts this signaling pathway.Results:A DNA fragment encoding the extracellular domain of CD40 (CD40-N) has been codon-optimized and cloned into pPIC9K to create a Pichia pastoris expression and secretion strain. SDS-PAGE and Western blotting assays using the culture media from methanol-induced expression strains showed that recombinant CD40-N, a 27 kDa glycosylated protein, was secreted into the culture broth. The recombinant protein was purified to more than 90 % using Sephadex G-50 size-exclusion chromatography and Q Sepharose Fast Flow ion exchange. Finally, 120 mg of the protein was obtained at a relatively high purity from 3 l supernatant. Binding assay (ITC200 assay) shown the direct interaction of CD40-N and CD40 agonist antibody (G28-5). The bioactivity of recombinant CD40-N was confirmed by its ability to disrupt non-canonical NF-κB signaling activated by CD40 agonist antibody or CD40 ligand and to inhibit ant-CD40 agonist antibody-induced TNF-alpha expression in BJAB cells in vitro. In addition, our data indicate that the protein has curative potential in treating dextran sulfate sodium (DSS)-induced colitis in vivo.Conclusions:The results show that the experimental procedure we have developed using P. pastoris can be used to produce large amounts of active CD40-N for research and industrial purposes. The protein fragment we have acquired has potential to be used in research or even treating inflammation diseases such as colitis.