• 主题
高级搜索  >
历史搜索 清空历史
首页> 美国卫生研究院文献>Wiley-Blackwell Online Open >Cell-free eukaryotic systems for the production engineering and modification of scFv antibody fragments
【2h】

Cell-free eukaryotic systems for the production engineering and modification of scFv antibody fragments

机译：用于scFv抗体片段生产工程设计和修饰的无细胞真核系统

代理获取
本网站仅为用户提供外文OA文献查询和代理获取服务，本网站没有原文。下单后我们将采用程序或人工为您竭诚获取高质量的原文，但由于OA文献来源多样且变更频繁，仍可能出现获取不到、文献不完整或与标题不符等情况，如果获取不到我们将提供退款服务。请知悉。
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Open cell-free translation systems based on Escherichia coli cell lysates have successfully been used to produce antibodies and antibody fragments. In this study, we demonstrate the cell-free expression of functional single-chain antibody variable fragments (scFvs) in a eukaryotic and endotoxin-free in vitro translation system based on Spodoptera frugiperda (Sf21) insect cell extracts. Three scFv candidates with different specificities were chosen as models. The first scFv candidate SH527-IIA4 specifically discriminates between its phosphorylated (SMAD2-P) and nonphosphorylated antigens (SMAD2) (where SMAD is mothers against decapentaplegic homolog 2), whereas the second scFv candidate SH527-IIC10 recognizes both, SMAD2-P and SMAD2. The third scFv candidate SH855-C11 binds specifically to a linear epitope of the CXC chemokine receptor type 5. The translocation of antibody fragments into the lumen of endogenous microsomal vesicles, which are contained in the lysate, was facilitated by fusion of scFv genes to the insect cell specific signal sequence of honeybee melittin. We compared the binding capabilities of scFv fragments with and without melittin signal peptide and detected that translocated scFv fragments were highly functional, whereas scFvs synthesized in the cytosol of the cell extract showed strongly decreased binding capabilities. Additionally, we describe a cell-free protein synthesis method for the incorporation of noncanonical amino acids into scFv molecules in eukaryotic cell lysates. We demonstrate the successful cotranslational labeling of de novo synthesized scFv molecules with fluorescent amino acids, using residue-specific as well as site-specific labeling.
机译：基于大肠杆菌细胞裂解物的无细胞无翻译系统已成功用于产生抗体和抗体片段。在这项研究中，我们证明了功能性单链抗体可变片段（scFvs）在基于贪夜蛾（Sf21）昆虫细胞提取物的真核和无内毒素体外翻译系统中的无细胞表达。选择具有不同特异性的三个scFv候选物作为模型。第一个scFv候选SH527-IIA4特异性区分其磷酸化（SMAD2-P）和非磷酸化抗原（SMAD2）（其中SMAD是抗十足功能同系物2的母亲），而第二个scFv候选SH527-IIC10识别SMAD2-P和SMAD2。 。第三种scFv候选蛋白SH855-C11与5型CXC趋化因子受体的线性表位特异性结合。scFv基因与CXC趋化因子受体5的线性表位特异性结合。抗体片段向裂解物中所含内源性微粒体囊泡的内腔转运。蜜蜂蜂毒素的昆虫细胞特异性信号序列。我们比较了具有和不具有蜂毒肽信号肽的scFv片段的结合能力，并检测到易位的scFv片段具有很高的功能，而在细胞提取物的胞质溶胶中合成的scFvs显示出结合能力大大降低。此外，我们描述了一种用于将非规范氨基酸掺入真核细胞裂解物中的scFv分子的无细胞蛋白质合成方法。我们证明了使用残基特异性和位点特异性标记成功地从头合成了带有荧光氨基酸的scFv分子的共翻译标记。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
代理获取

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号