Abstract：
High-throughput assays that efficiently link genotype and phenotype with high fidelity are key to successful enzyme engineering campaigns. Among these assays, the tyramide/peroxidase proximity labeling method converts the product of an enzymatic reaction of a surface expressed enzyme to a highly reactive fluorescent radical, which labels the cell surface. In this context, maintaining the proximity of the readout reagents to the cell surface is crucial to prevent crosstalk and ensure that short-lived radical species react before diffusing away. Here, we investigated improvements in tyramide/peroxidase proximity labeling for enzyme screening. We modified chitosan (Cs) chains with horseradish peroxidase (HRP) and evaluated the effects of these conjugates on the efficiency of proximity labeling reactions on yeast cells displaying d-amino acid oxidase. By tethering HRP to chitosan through different chemical approaches, we localized the auxiliary enzyme close to the cell surface and enhanced the sensitivity of tyramide-peroxidase labeling reactions. We found that immobilizing HRP onto chitosan through a 5 kDa PEG linker improved labeling sensitivity by over 3.5-fold for substrates processed with a low turnover rate (e.g., d-lysine), while the sensitivity of the labeling for high activity substrates (e.g., d-alanine) was enhanced by over 0.6-fold. Such improvements in labeling efficiency broaden the range of enzymes and conditions that can be studied and screened by tyramide/peroxidase proximity labeling.
摘要：
高效地将基因型和表型以高保真度联系起来的高通量测定是成功的酶工程活动的关键。在这些检测中,酪胺/过氧化物酶邻近标记方法将表面表达酶的酶反应产物转化为高反应性荧光自由基，标记细胞表面。在这种情况下，保持读出试剂与细胞表面的接近度对于防止串扰和确保短寿命的自由基物种在扩散离开之前反应是至关重要的。这里,我们研究了酪胺/过氧化物酶邻近标记用于酶筛选的改进。我们用辣根过氧化物酶（HRP）修饰了壳聚糖（Cs）链，并评估了这些缀合物对显示d-氨基酸氧化酶的酵母细胞的邻近标记反应效率的影响。通过不同的化学方法将HRP连接到壳聚糖上，我们将辅助酶定位在细胞表面附近，并增强了酪胺-过氧化物酶标记反应的敏感性。我们发现，通过5kDaPEG接头将HRP固定到壳聚糖上，对于以低周转率处理的底物，标记灵敏度提高了3.5倍以上（例如，d-赖氨酸)，而标记对高活性底物的敏感性(例如，d-丙氨酸）增加了0.6倍以上。标记效率的这种改进拓宽了可通过酪胺/过氧化物酶邻近标记研究和筛选的酶和条件的范围。
