Purification of SARS-CoV-2 RBD in Affinity Chromatography Using a Novel Nanobody Ligand. undefined

In the spike protein of SARS-CoV-2, the receptor-binding domain (RBD) contains multiple dominant neutralizing epitopes and can be used as an antigen for developing COVID-19 vaccines and neutral antibodies. Affinity chromatography is one of the most extensively used methods for rapid one-step protein purification. However, there is a lack of commercially available affinity ligands for RBD purification. Here, we report the rapid isolation of a nanobody suitable for purifying RBD as an affinity ligand from immune phage display libraries. After bio-panning, the enriched clones were sequenced on next-generation sequencing (NGS) platforms and classified into four groups based on the CDRH3 amino acid sequence. The representative sequences with high nanomolar affinities to RBD were further categorized into two groups via epitope binning analysis. Finally, from the two epitope bins, we found that SS3 showed easy elution under a mild eluting condition and could be used as a functional affinity ligand to purify RBD. These results also indicate that categorizing the bio-panned sequences via high-throughput sequencing (HTS) techniques followed by epitope binning represents a fast workflow to select specific binders with desired properties. Keywords: phage display; nanobody; Affinity chromatography; epitope binning, NGS sequence

在严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）的刺突蛋白中，受体结合域（receptor-binding domain, RBD）包含多个优势中和表位，可作为开发新型冠状病毒肺炎（COVID-19）疫苗和中和抗体的抗原。
亲和层析是目前应用最为广泛的快速一步式蛋白质纯化技术之一，但暂无商用的可用于RBD纯化的亲和配体。
本研究从免疫噬菌体展示文库中快速分离得到可作为亲和配体用于RBD纯化的纳米抗体（nanobody）。
经生物淘选（bio-panning）后，通过下一代测序（next-generation sequencing, NGS）平台对富集的克隆进行测序，并基于重链互补决定区3（CDRH3）的氨基酸序列将其分为4组。
对与RBD具有高纳摩尔级亲和力的代表性序列，通过表位分组分析（epitope binning analysis）进一步分为2组。
最终，从这2个表位分组中筛选得到SS3，其可在温和洗脱条件下易于洗脱，可作为功能性亲和配体用于RBD的纯化。
本研究结果同时表明，通过高通量测序（high-throughput sequencing, HTS）技术对生物淘选得到的序列进行分类，再结合表位分组分析，是一种可快速筛选获得具备目标特性特异性结合剂的高效流程。
关键词：噬菌体展示；纳米抗体；亲和层析；表位分组；NGS序列