Department of Chemical and Biomolecular Engineering, University of Connecticut, Storrs, CT 06269, USA.
Department of Biomedical Engineering, University of Connecticut, Storrs, CT 06269, USA.
Protein Eng Des Sel. 2023 Jan 21;36. doi: 10.1093/protein/gzad005.
The detection of site-specific phosphorylation in the microtubule-associated protein tau is emerging as a means to diagnose and monitor the progression of Alzheimer's Disease and other neurodegenerative diseases. However, there is a lack of phospho-specific monoclonal antibodies and limited validation of their binding specificity. Here, we report a novel approach using yeast biopanning against synthetic peptides containing site-specific phosphorylations. Using yeast cells displaying a previously validated phospho-tau (p-tau) single-chain variable region fragment (scFv), we show selective yeast cell binding based on single amino acid phosphorylation on the antigen. We identify conditions that allow phospho-specific biopanning using scFvs with a wide range of affinities (KD = 0.2 to 60 nM). Finally, we demonstrate the capability of screening large libraries by performing biopanning in 6-well plates. These results show that biopanning can effectively select yeast cells based on phospho-site specific antibody binding, opening doors for the facile identification of high-quality monoclonal antibodies.
检测微管相关蛋白 tau 中的特定磷酸化位点正成为诊断和监测阿尔茨海默病和其他神经退行性疾病进展的一种手段。然而，目前缺乏磷酸化特异性单克隆抗体，并且其结合特异性的验证也很有限。在这里，我们报告了一种使用酵母生物淘选针对含有特定磷酸化位点的合成肽的新方法。我们使用展示先前经过验证的磷酸化 tau (p-tau) 单链可变区片段 (scFv) 的酵母细胞，根据抗原上单个氨基酸的磷酸化显示出选择性的酵母细胞结合。我们确定了在具有广泛亲和力 (KD = 0.2 至 60 nM) 的 scFv 条件下允许进行磷酸化特异性生物淘选的条件。最后，我们通过在 6 孔板中进行生物淘选证明了筛选大型文库的能力。这些结果表明，生物淘选可以有效地根据磷酸化位点特异性抗体结合来选择酵母细胞，为方便鉴定高质量的单克隆抗体开辟了道路。
