Antonescu Oana N, Rasmussen Andreas, Damm Nicole A M, Heidemann Ditte F, Popov Roman, Nesterov-Mueller Alexander, Johansson Kristoffer E, Winther Jakob R
Department of Biology, Linderstrøm-Lang Centre for Protein Science, Section for Biomolecular Sciences, University for Copenhagen, Copenhagen, Denmark.
Institute of Microstructure Technology, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen, Germany.
PLoS One. 2021 Feb 3;16(2):e0241461. doi: 10.1371/journal.pone.0241461. eCollection 2021.
Split fluorescent proteins have wide applicability as biosensors for protein-protein interactions, genetically encoded tags for protein detection and localization, as well as fusion partners in super-resolution microscopy. We have here established and validated a novel platform for functional analysis of leave-one-out split fluorescent proteins (LOO-FPs) in high throughput and with rapid turnover. We have screened more than 12,000 variants of the beta-strand split fragment using high-density peptide microarrays for binding and functional complementation in Green Fluorescent Protein. We studied the effect of peptide length and the effect of different linkers to the solid support. We further mapped the effect of all possible amino acid substitutions on each position as well as in the context of some single and double amino acid substitutions. As all peptides were tested in 12 duplicates, the analysis rests on a firm statistical basis allowing for confirmation of the robustness and precision of the method. Based on experiments in solution, we conclude that under the given conditions, the signal intensity on the peptide microarray faithfully reflects the binding affinity between the split fragments. With this, we are able to identify a peptide with 9-fold higher affinity than the starting peptide.
分裂荧光蛋白作为蛋白质-蛋白质相互作用的生物传感器、用于蛋白质检测和定位的基因编码标签以及超分辨率显微镜中的融合伙伴具有广泛的适用性。我们在此建立并验证了一个新平台，用于高通量且快速周转地对逐个缺失分裂荧光蛋白（LOO-FP）进行功能分析。我们使用高密度肽微阵列筛选了超过12000个β链分裂片段变体，以检测其在绿色荧光蛋白中的结合和功能互补情况。我们研究了肽长度的影响以及不同连接子对固相支持物的影响。我们进一步绘制了每个位置上所有可能氨基酸取代的影响，以及一些单氨基酸和双氨基酸取代情况下的影响。由于所有肽都进行了12次重复测试，该分析基于坚实的统计基础，从而能够确认该方法的稳健性和精确性。基于溶液中的实验，我们得出结论，在给定条件下，肽微阵列上的信号强度忠实地反映了分裂片段之间的结合亲和力。据此，我们能够鉴定出一种亲和力比起始肽高9倍的肽。
