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.

中文翻译：

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用于生产、改造和修饰 scFv 抗体片段的无细胞真核系统

基于大肠杆菌细胞裂解物的开放式无细胞翻译系统已成功用于生产抗体和抗体片段。在这项研究中，我们证明了功能性单链抗体可变片段（scFvs）在基于草地贪夜蛾（Sf21）昆虫细胞提取物的真核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 候选者作为模型。第一个候选 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 是针对十五位麻痹同源物 2 的母亲），而第二个候选 scFv 候选 SH527-IIC10 识别 SMAD2-P 和 SMAD2 . 第三个候选 scFv 的 SH855-C11 与 CXC 5 型趋化因子受体的线性表位特异性结合。scFv 基因与蜜蜂蜂毒肽的昆虫细胞特异性信号序列融合促进了抗体片段向内源性微粒体囊泡腔内的易位，这些囊泡包含在裂解物中。我们比较了有和没有蜂毒肽信号肽的 scFv 片段的结合能力，并检测到易位的 scFv 片段具有高度功能，而在细胞提取物的胞质溶胶中合成的 scFv 显示出强烈降低的结合能力。此外，我们描述了一种无细胞蛋白质合成方法，用于将非经典氨基酸掺入真核细胞裂解物中的 scFv 分子中。我们证明了使用残基特异性和位点特异性标记成功地用荧光氨基酸对从头合成的 scFv 分子进行共翻译标记。