Preparation and characterization of a novel exendin-4 human serum albumin fusion protein expressed in Pichia pastoris
Preparation and characterization of a novel exendin-4 human serum albumin fusion protein expressed in Pichia pastoris
复制标题
巴斯德毕赤酵母表达的新型exendin-4人血清清蛋白融合蛋白的制备及表征
DOI：
10.1002/psc.942
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发表时间：
2008-05-01
影响因子：
2.1
通讯作者：
Zhou, Lin-Fu
中科院分区：
文献类型：
--
作者：
Huang, Yan-Shan;Chen, Zhi;Zhou, Lin-Fu
利用毕赤酵母（Pichia pastoris）表达了一种新的重组exendin-4人血清白蛋白融合蛋白（雷克斯-4/HSA）。Ex-4是从蜥蜴Heloderma suspectum的唾液腺中分离出的一种39个氨基酸的肽，被认为是2型糖尿病的新型治疗药物。但是为了获得持续的效果，由于其短的血浆半衰期（T-1/2 = 2.4 h），必须每天注射两次肽。为了延长Ex-4分子在体内的半衰期，我们设计了基因工程Ex-4/HSA融合蛋白。在Ex-4和HSA之间插入肽接头GGGGS，并在具有天然HSA分泌信号序列的甲基营养型酵母中表达融合蛋白。重组蛋白被正确分泌，并通过三步纯化程序以高纯度（通常>98%）获得。cAMP测定表明，该融合蛋白在体外与GLP-1受体的相互作用具有与Ex-4相似的生物活性。口服葡萄糖耐量试验结果表明，雷克斯-4/HSA能有效改善db/db糖尿病小鼠的糖耐量。食蟹猴中的药代动力学研究也表明，雷克斯-4/HSA具有更长的血浆半衰期。因此，雷克斯-4/HSA融合蛋白有可能成为治疗2型糖尿病的新型重组生物药物。版权所有（C）2007欧洲肽协会和约翰威利父子有限公司。
A novel recombinant exendin-4 human serum albumin fusion protein (rEx-4/HSA) expressed in Pichia pastoris was prepared and characterized. Ex-4 is a 39-amino acid peptide isolated from the salivary gland of the lizard Heloderma suspectum and is thought to be a novel therapeutic agent for type 2 diabetes. But to gain a continued effect, the peptide has to be injected twice a day owing to its short plasma half-life (T-1/2 = 2.4 h). To extend the half-life of Ex-4 molecule in vivo, we designed a genetically engineered Ex-4/HSA fusion protein. Between Ex-4 and HSA, a peptide linker GGGGS was inserted and the fusion protein was expressed in methylotrophic yeast P. pastoris with native HSA secretion signal sequence. The recombinant protein was secreted correctly and was obtained with high purity (typically >98%) by a three-step purification procedure. cAMP assay demonstrated that the fusion protein had a bioactivity similar to Ex-4 for interaction with GLP-1 receptors in vitro. Results from oral glucose tolerance test indicated that rEx-4/HSA could effectively improve glucose tolerance in diabetic db/db mice. Pharmacokinetics studies in cynomologus monkeys also showed that rEx-4/HSA had a much longer plasma half-life. Therefore, rEx-4/HSA fusion protein could potentially be used as a new recombinant biodrug for type 2 diabetes therapy. Copyright (C) 2007 European Peptide Society and John Wiley & Sons, Ltd.