Improving protein pharmacokinetics by genetic fusion to simple amino acid sequences
Improving protein pharmacokinetics by genetic fusion to simple amino acid sequences
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DOI：
10.1074/jbc.m311356200
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发表时间：
2004-01-30
影响因子：
4.8
通讯作者：
Campetella, O
中科院分区：
文献类型：
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作者：
Alvarez, P;Buscaglia, CA;Campetella, O
一级氨基酸序列在蛋白质药代动力学中的作用，在基础知识和生物技术中的相关问题，在这里作为起点使用两个重复的抗原血鞭毛虫克氏锥虫，已知稳定其相关的蛋白质在血流中。它们的药理学应用的主要缺点是这些重复序列是高度免疫原性的，因此需要缺失该特征。基于序列同源性和表位作图分析，设计了人工重复序列（PSTAD）。该基序通过与锥虫转唾液酸酶和大鼠酪氨酸氨基转移酶的C末端的基因融合进行测试，发现在血液中相关蛋白的半衰期增加4.5-6倍，同时显示出显著较低的免疫原性。通过定点诱变方法绘制了参与新肽稳定特性的残基，使我们能够成功地识别另外两个基序。搜索数据库中显示一些同源性的序列，嵌入在脯氨酸框架和相关的脱落的毒力因子从无关的微生物，导致在其他四个蛋白质的延伸的鉴定。值得注意的是，其中三种（来自肺炎链球菌，粘性放线菌和大肠杆菌）显示出相似的药代动力学特征，因此表明类似的进化获得机制，以确保其相应蛋白质的生物分布。我们的研究结果表明，插入到一个富含脯氨酸的框架定义的基序构成了一个合适的替代构建嵌合蛋白在血液中具有延长的半衰期。
The role of primary amino acid sequences in protein pharmacokinetics, an issue of relevance in both basic knowledge and biotechnology, was addressed here using as a starting point two repetitive antigens from the hemoflagellate Trypanosoma cruzi that are known to stabilize their associated proteins in the bloodstream. A major drawback to their pharmacological application is that these repetitive sequences are highly immunogenic, being therefore the deletion of this characteristic desirable. Based on sequence homology and epitope mapping analyses, an artificial repetitive sequence (PSTAD) was engineered. This motif was tested by genetic fusion to the C terminus of both the trypanosomal trans-sialidase and the rat tyrosine aminotransferase and found to produce a 4.5-6-fold increase in the half-life of the associated proteins in blood while displaying significantly lower immunogenicity. Residues involved in the stabilizing properties of the novel peptide were mapped by a site-directed mutagenesis approach, allowing us to successfully identify another two motifs. Searching databases for sequences displaying some homology, embedded in proline frameworks and associated to shed virulence factors from unrelated microorganisms, resulted in the identification of four other protein extensions. Remarkably, three of them (from Streptococcus pneumoniae, Actinomyces viscosus, and Escherichia coli) revealed similar pharmacokinetic features, suggesting therefore an analogous evolutionarily acquired mechanism to ensure the biodistribution of their corresponding proteins. Our findings indicate that the insertion of defined motifs into a proline-rich framework constitutes a suitable alternative to construct a chimeric protein with extended half-life in blood.