重组人白细胞介素-4在毕赤酵母中的表达及其糖基化与生物学活性的关系。
Expression of recombinant human IL-4 in Pichia pastoris and relationship between its glycosylation and biological activity.
作者信息
Li Rui, Xie Chao, Zhang Yuan, Li Bin, Donelan William, Li Shiwu, Han Shuhong, Wang Xingli, Cui Taixing, Tang Dongqi
机构信息
Center for Stem Cell & Regenerative Medicine, The Second Hospital of Shandong University, Jinan 250012, PR China; Shandong University Qilu Hospital Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital of Shandong University, Jinan 250012, PR China.
Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA; College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China.
出版信息
Protein Expr Purif. 2014 Apr;96:1-7. doi: 10.1016/j.pep.2014.01.005. Epub 2014 Jan 24.
Secretory human interleukin 4 (hIL4) is an N-glycosylated pleiotropic cytokine. It is unknown if these N-linked glycans are required and essential for hIL4 protein stability, expression, secretion, and activity in vivo, and hIL4 expressed from Pichia pastoris yeast has not been tested to date. In this study, we successfully expressed human hIL4 in P. pastoris, the methylotrophic yeast, with a yield of 15.0mg/L. Using the site-directed mutagenesis technique, we made two mutant hIL4 cDNA clones (N38A and N105L) and subsequently expressed them in P. pastoris to analyze the relevant function of each N-glycosylation site on hIL4. Our results demonstrate that the glycosylation only occurs at position Asn38, but not Asn105. The glycosylated form of hIL4 unexpectedly has lower biological activity and lower stability when compared to its non-glycosylated form. The implications of this are discussed.
分泌型人白细胞介素4（hIL4）是一种N-糖基化的多效性细胞因子。目前尚不清楚这些N-连接聚糖对于hIL4蛋白在体内的稳定性、表达、分泌和活性是否是必需的，且迄今尚未对毕赤酵母表达的hIL4进行测试。在本研究中，我们成功地在甲基营养型酵母毕赤酵母中表达了人hIL4，产量为15.0mg/L。利用定点诱变技术，我们制备了两个突变型hIL4 cDNA克隆（N38A和N105L），随后在毕赤酵母中表达它们，以分析hIL4上每个N-糖基化位点的相关功能。我们的结果表明，糖基化仅发生在Asn38位点，而不是Asn105位点。与非糖基化形式相比，hIL4的糖基化形式意外地具有较低的生物活性和较低的稳定性。本文对其意义进行了讨论。
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