Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, United States of America.
Specifica Inc., Santa Fe, NM, United States of America.
PLoS One. 2023 Feb 24;18(2):e0280930. doi: 10.1371/journal.pone.0280930. eCollection 2023.
Antibodies are important reagents for research, diagnostics, and therapeutics. Many examples of chimeric proteins combining the specific target recognition of antibodies with complementing functionalities such as fluorescence, toxicity or enzymatic activity have been described. However, antibodies selected solely on the basis of their binding specificities are not necessarily ideal candidates for the construction of chimeras. Here, we describe a high throughput method based on yeast display to directly select antibodies most suitable for conversion to fluorescent chimera. A library of scFv binders was converted to a fluorescent chimeric form, by cloning thermal green protein into the linker between VH and VL, and directly selecting for both binding and fluorescent functionality. This allowed us to directly identify antibodies functional in the single chain TGP format, that manifest higher protein expression, easier protein purification, and one-step binding assays.
抗体是研究、诊断和治疗的重要试剂。已经描述了许多将抗体的特异性靶标识别与互补功能（如荧光、毒性或酶活性）结合在一起的嵌合蛋白的例子。然而，仅基于结合特异性选择的抗体不一定是构建嵌合体的理想候选物。在这里，我们描述了一种基于酵母展示的高通量方法，可直接选择最适合转化为荧光嵌合体的抗体。通过将热绿色蛋白克隆到 VH 和 VL 之间的连接子中，将 scFv 结合物文库转化为荧光嵌合形式，并直接选择结合和荧光功能。这使我们能够直接识别在单链 TGP 形式下具有功能的抗体，这些抗体表现出更高的蛋白表达量、更简单的蛋白纯化和一步结合测定。
