经改良的 Sortase 自我切割-细菌表面展示系统产生的针对病毒 RNA 依赖性 RNA 聚合酶的人源超级抗体。

Human super antibody to viral RNA-dependent RNA polymerase produced by a modified Sortase self-cleave-bacteria surface display system.

机构信息

Center of Research Excellence in Therapeutic Proteins and Antibody Engineering, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand.

School of Pathology, Translational Medicine Program, Institute of Medicine, Suranaree University of Technology, Nakhon Ratchasima, 30000, Thailand.

出版信息

Microb Cell Fact. 2023 Dec 18;22(1):260. doi: 10.1186/s12934-023-02267-z.

DOI:10.1186/s12934-023-02267-z

PMID:38110987

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10726597/

Abstract

BACKGROUND

RNA-dependent RNA polymerase (RdRp) is a good target of anti-RNA virus agents; not only it is pivotal for the RNA virus replication cycle and highly conserved among RNA viruses across different families, but also lacks human homolog. Recently, human single-chain antibody (HuscFv) that bound to thumb domain of hepatitis C virus (HCV) RNA-dependent RNA polymerase (functionalized NS5B protein) was produced and engineered into cell-penetrating antibody (super antibody) in the form of cell-penetrating peptide (penetratin, PEN)-linked HuscFv (PEN-HuscFv34). The super antibody was produced and purified from inclusion body (IB) of a pen-huscfv34-vector-transformed Escherichia coli. The super antibody inhibited replication of alpha- and beta- coronaviruses, flaviviruses, and picornaviruses that were tested (broadly effective); thus, it has high potential for developing further towards a pan-anti-RNA virus agent. However, production, purification, and refolding of the super antibody molecules from the bacterial IB are laborious and hurdles to large-scale production. Therefore, in this study, Sortase-self-cleave method and bacteria surface display system were combined and modified for the super antibody production.

METHODS AND RESULTS

BL21 (DE3) ΔA E. coli, a strain lacking predominant outer membrane protein (OmpA) and ion and OmpT proteases, that displayed a membrane-anchored fusion protein, i.e., chimeric lipoprotein (Lpp')-OmpA', SUMO, Sortase protease, Sortase cleavage site (LPET↓G) and PEN-HuscFv34-6× His was generated. The soluble PEN-HuscFv34-6× His with glycine at the N-terminus could be released from the E. coli surface, simply by incubating the bacterial cells in a Sortase-cleavage buffer. After centrifugation, the G-PEN-HuscFv34-6× His could be purified from the supernatant. The purified G-PEN-HuscFv34-6× retained original cell-penetrating ability (being super antibody) and the broadly effective anti-RNA virus activity of the original IB-derived-PEN-HuscFv34.

CONCLUSION

The functionalized super antibody to RNA virus RdRp was successfully produced by using combined Sortase self-cleave and bacterial surface display systems with modification. The display system is suitable for downstream processing in a large-scale production of the super antibody. It is applicable also for production of other recombinant proteins in soluble free-folding form.

摘要

背景

RNA 依赖的 RNA 聚合酶（RdRp）是抗 RNA 病毒药物的一个很好的靶点；它不仅是 RNA 病毒复制周期的关键，而且在不同科的 RNA 病毒中高度保守，并且缺乏人类同源物。最近，生产了与人 HCV RNA 依赖性 RNA 聚合酶（功能性 NS5B 蛋白）结合的单链抗体（HuscFv），并将其工程化为穿透细胞的抗体（超级抗体）的形式穿透肽（ penetratin，PEN）-连接的 HuscFv（PEN-HuscFv34）。超级抗体由 PEN-huscfv34-载体转化的大肠杆菌包涵体（IB）中产生和纯化。超级抗体抑制了所测试的α和β冠状病毒，黄病毒和小核糖核酸病毒的复制（广泛有效）；因此，它具有进一步开发成为泛抗 RNA 病毒药物的巨大潜力。但是，从细菌 IB 中生产，纯化和重折叠超级抗体分子是费力的，并且是大规模生产的障碍。因此，在这项研究中，组合和修饰了 Sortase 自身切割方法和细菌表面展示系统，以生产超级抗体。

方法和结果

BL21（DE3）ΔA 大肠杆菌，一种缺乏主要外膜蛋白（OmpA）和离子和 OmpT 蛋白酶的菌株，展示了一种膜锚定的融合蛋白，即嵌合脂蛋白（Lpp'）-OmpA'，SUMO，Sortase 蛋白酶，Sortase 切割位点（LPET↓G）和 PEN-HuscFv34-6×His。N 末端带有甘氨酸的可溶性 PEN-HuscFv34-6×His 可以通过简单地将细菌细胞在 Sortase 切割缓冲液中孵育，从大肠杆菌表面释放出来。离心后，可从上清液中纯化 G-PEN-HuscFv34-6×His。纯化的 G-PEN-HuscFv34-6×His 保留了原始 PEN-HuscFv34-6×His 的穿透细胞能力（为超级抗体）和原始 IB 衍生的 PEN-HuscFv34 的广泛有效的抗 RNA 病毒活性。