Sun Yudong, Martinez-Ramos Cristian, Chen Eugene, Osawa Yoichi, Zhang Haoming
Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Internal Medicine, The University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Biomedicines. 2024 Aug 15;12(8):1863. doi: 10.3390/biomedicines12081863.
The availability of yeast surface display nanobody (Nb) libraries offers a convenient way to acquire antigen-specific nanobodies that may be useful for protein structure-function studies and/or therapeutic applications, complementary to the conventional method of acquiring nanobodies through immunization in camelids. In this study, we developed a general approach to select nanobodies for cytochrome P450 enzymes from a highly diverse yeast display library. We tested our method on three P450 enzymes including CYP102A1, neuronal nitric oxide synthase (nNOS), and the complex of CYP2B4:POR, using a novel streamlined approach where biotinylated P450s were bound to fluorescent-labeled streptavidin for Nb screening. The Nb-antigen binders were selectively enriched using magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). After two rounds of MACS, the population of positive binders was enriched by >5-fold compared to the naïve library. The subsequent FACS selection, with a gating of 0.1%, identified 634, 270, and 215 positive binders for CYP102A1, nNOS, and CYP2B4:POR, respectively. The positive binders for CYP102A1 were further triaged based on EC determined at various antigen concentrations. DNA sequencing of the top 30 binders of CYP102A1 resulted in 26 unique clones, 8 of which were selected for over-expression and characterization. They were found to inhibit CYP102A1-catalyzed oxidation of omeprazole with IC values in the range of 0.16-2.8 µM. These results validate our approach and may be applied to other protein targets for the effective selection of specific nanobodies.
酵母表面展示纳米抗体（Nb）文库的出现，为获取抗原特异性纳米抗体提供了一种便捷的方法，这对于蛋白质结构功能研究和/或治疗应用可能是有用的，是对通过骆驼科动物免疫获取纳米抗体的传统方法的补充。在本研究中，我们开发了一种通用方法，用于从高度多样化的酵母展示文库中筛选细胞色素P450酶的纳米抗体。我们使用一种新颖的简化方法，将生物素化的P450与荧光标记的链霉亲和素结合用于Nb筛选，在三种P450酶上测试了我们的方法，这三种酶包括CYP102A1、神经元型一氧化氮合酶（nNOS）以及CYP2B4:POR复合物。使用磁珠激活细胞分选（MACS）和荧光激活细胞分选（FACS）选择性富集Nb-抗原结合物。经过两轮MACS，与原始文库相比，阳性结合物群体富集了5倍以上。随后的FACS筛选，门控为0.1%，分别鉴定出CYP102A1、nNOS和CYP2B4:POR的634、270和215个阳性结合物。基于在各种抗原浓度下测定的EC对CYP102A1的阳性结合物进行进一步分类。对CYP102A1的前30个结合物进行DNA测序，得到26个独特克隆，其中8个被选择用于过表达和表征。发现它们抑制CYP102A1催化的奥美拉唑氧化，IC值在0.16-2.8μM范围内。这些结果验证了我们的方法，并且可能适用于其他蛋白质靶点，以有效筛选特异性纳米抗体。
