Nielsen Gregory H, Sachs Jonathan N, Hackel Benjamin J
Department of Chemical Engineering and Materials Science, University of Minnesota Twin Cities, Minneapolis, Minnesota, USA.
Department of Biomedical Engineering, University of Minnesota Twin Cities, Minneapolis, Minnesota, USA.
Biotechnol Bioeng. 2025 Jun;122(6):1386-1396. doi: 10.1002/bit.28954. Epub 2025 Mar 5.
Protein developability is an important, yet often overlooked, aspect of protein discovery campaigns that is a key driver of utility. Recent advances have improved developability screening capacity, making it an increasingly viable option in early-stage discovery. Here, we engineered one component of developability, stability, of two affibody proteins-one that targets death receptor 5 and another that targets tumor necrosis factor receptor 1-previously evolved to bind receptor and non-competitively inhibit signaling via conformational modulation. Starting from an error-prone PCR library of each affibody, variants were screened via yeast surface display binder selections, including depletion of non-specific binders, followed by developability assessment using the on-yeast protease and yeast display level assays. Multiplex deep sequencing identified variants for further evaluation. Purified variants exhibited elevated stability-8°C to 14°C increase in T-with maintained 1-2 nM affinity for the TNFR1 affibody and 30-fold improvement in the DR5 affibody affinity to 0.8 nM.
蛋白质可开发性是蛋白质发现活动中一个重要但常被忽视的方面，是实用性的关键驱动因素。最近的进展提高了可开发性筛选能力，使其在早期发现中成为越来越可行的选择。在此，我们对两种亲和体蛋白的可开发性的一个组成部分——稳定性进行了工程改造，一种亲和体蛋白靶向死亡受体5，另一种靶向肿瘤坏死因子受体1，这两种亲和体蛋白先前经过进化以结合受体并通过构象调节非竞争性抑制信号传导。从每个亲和体的易错PCR文库开始，通过酵母表面展示结合物筛选来筛选变体，包括去除非特异性结合物，然后使用酵母上蛋白酶和酵母展示水平测定法进行可开发性评估。多重深度测序确定了用于进一步评估的变体。纯化后的变体表现出更高的稳定性——熔解温度（Tm）提高了8°C至14°C，对TNFR1亲和体的亲和力维持在1 - 2 nM，DR5亲和体的亲和力提高了30倍，达到0.8 nM。
