[Cloning and recombinant expression of human soluble TRAIL in Pichia pastoris].
[Cloning and recombinant expression of human soluble TRAIL in Pichia pastoris].
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发表时间：
2010-12
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通讯作者：
Yingying Li;L. Wan;Hao Yang;Shan Liu;H. Cai;Xiaofeng Lu
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作者：
Yingying Li;L. Wan;Hao Yang;Shan Liu;H. Cai;Xiaofeng Lu
肿瘤坏死因子相关细胞凋亡诱导配体（TNF-related apoptosis-inducing ligand，TRAIL）是肿瘤坏死因子家族的成员之一，具有选择性、高效诱导肿瘤细胞凋亡的作用，有望开发为新型抗肿瘤药物。基因重组表达是生产药用蛋白的重要手段。本研究利用PCR技术克隆了人可溶性TRAIL基因（114- 281 aa片段），并将其克隆到毕赤酵母表达载体pPIC 9 K中。在含有新霉素G418的平板上对转化体进行双重筛选，并选择具有高水平G418抗性的许多克隆用于蛋白表达的进一步研究。重组人可溶性TRAIL在3%甲醇条件下分泌到BMMY培养基中。重组蛋白经Ni-琼脂糖亲和层析纯化至纯度约80%。这种蛋白质的产量约为每升培养物1-2毫克。细胞活力测定表明，人可溶性TRAIL对白血病细胞Jurkat和肺癌细胞A549都具有细胞毒性。经0.05 μ g/ml TRAIL处理后，Jurkat细胞的存活率约为10%。表达的TRAIL在0.1-1 μ g/ml范围内对A549细胞显示出剂量依赖性的细胞毒性。当蛋白浓度达到1 μ g/ml时，A549细胞的存活率约为30%。然而，重组人可溶性TRAIL在所测试的浓度下在人皮肤成纤维细胞（HSF）中未显示出明显的细胞毒性。这些结果表明，人可溶性TRAIL在肿瘤细胞中具有选择性细胞毒性。本实验构建的表达系统为进一步大量生产可溶性TRAIL和基于TRAIL的新型融合蛋白奠定了基础。
TNF-related apoptosis-inducing ligand (TRAIL) is a member of factor TNF family, which could be potentially developed as novel antitumor agent due to its selective and efficient induction of apoptosis in tumor cells. Gene recombinant expression is an important tool for production of pharmaceutical protein. In this paper, the gene encoding human soluble TRAIL (114-281aa fragment) was cloned by PCR and then inserted into the Pichia Pastoris expression vector pPIC9K. The transformants were double-screened on plates containing neomycin G418 and many clones with high levels of G418-resistance were selected for further studies on protein expression. The recombinant human soluble TRAIL was secreted into the BMMY media under the condition of 3% methanol. And the recombinant protein was purified to homogeneity (-80% purity) by using Ni-agarose affinity chromatography. The yield of this protein is about 1-2 mg per liter culture. Cell viability assays demonstrated that human soluble TRAIL was cytotoxic in both leukemia cells Jurkat and lung cancer cells A549. After treatment with 0.05 microg/ml TRAIL, the survival rate of Jurkat cells was about 10%. The expressed TRAIL showed dose-dependent cytotoxicity in A549 cells within the range of 0.1-1 microg/ml. When the protein concentration reached 1 microg/ml, the survival rates of A549 cells were about 30%. However, the recombinant human soluble TRAIL did not show obvious cytotoxicity in human skin fibroblast cells (HSF) at concentrations tested. There results demonstrate that human soluble TRAIL is selectively cytotoxic in tumor cells. The expression system constructed in this experiment might contribute to further production of soluble TRAIL and TRAIL-based novel fusion proteins in large quantities.