Center for Metabolic Disease Research, Temple University Lewis Katz School of Medicine, Philadelphia, PA 19140, USA; Department of Pathology and Laboratory Medicine, Temple University Lewis Katz School of Medicine, Philadelphia, PA 19140, USA.
Department of Pathology and Laboratory Medicine, Temple University Lewis Katz School of Medicine, Philadelphia, PA 19140, USA.
Mol Ther. 2023 Apr 5;31(4):1136-1158. doi: 10.1016/j.ymthe.2023.02.012. Epub 2023 Feb 14.
Boosting protein production is invaluable in both industrial and academic applications. We discovered a novel expression-increasing 21-mer cis-regulatory motif (Exin21) that inserts between SARS-CoV-2 envelope (E) protein-encoding sequence and luciferase reporter gene. This unique Exin21 (CAACCGCGGTTCGCGGCCGCT), encoding a heptapeptide (QPRFAAA, designated as Qα), significantly (34-fold on average) boosted E production. Both synonymous and nonsynonymous mutations within Exin21 diminished its boosting capability, indicating the exclusive composition and order of 21 nucleotides. Further investigations demonstrated that Exin21/Qα addition could boost the production of multiple SARS-CoV-2 structural proteins (S, M, and N) and accessory proteins (NSP2, NSP16, and ORF3), and host cellular gene products such as IL-2, IFN-γ, ACE2, and NIBP. Exin21/Qα enhanced the packaging yield of S-containing pseudoviruses and standard lentivirus. Exin21/Qα addition on the heavy and light chains of human anti-SARS-CoV monoclonal antibody robustly increased antibody production. The extent of such boosting varied with protein types, cellular density/function, transfection efficiency, reporter dosage, secretion signaling, and 2A-mediated auto-cleaving efficiency. Mechanistically, Exin21/Qα increased mRNA synthesis/stability, and facilitated protein expression and secretion. These findings indicate that Exin21/Qα has the potential to be used as a universal booster for protein production, which is of importance for biomedicine research and development of bioproducts, drugs, and vaccines.
提高蛋白质产量在工业和学术应用中都具有重要价值。我们发现了一种新的表达增强 21 个核苷酸顺式调控元件（Exin21），它插入 SARS-CoV-2 包膜（E）蛋白编码序列和荧光素酶报告基因之间。这种独特的 Exin21（CAACCGCGGTTCGCGGCCGCT），编码一个七肽（QPRFAAA，命名为 Qα），可显著（平均 34 倍）增强 E 蛋白的产生。Exin21 中的同义和非同义突变都降低了其增强能力，表明 21 个核苷酸的组成和顺序是独特的。进一步的研究表明，Exin21/Qα 的添加可以增强多种 SARS-CoV-2 结构蛋白（S、M 和 N）和辅助蛋白（NSP2、NSP16 和 ORF3）以及宿主细胞基因产物（如 IL-2、IFN-γ、ACE2 和 NIBP）的产生。Exin21/Qα 增强了含有 S 蛋白的假病毒和标准慢病毒的包装产量。Exin21/Qα 添加到人抗 SARS-CoV 单克隆抗体的重链和轻链上，可显著增加抗体的产生。这种增强的程度因蛋白类型、细胞密度/功能、转染效率、报告基因剂量、分泌信号和 2A 介导的自身切割效率而异。从机制上讲，Exin21/Qα 增加了 mRNA 的合成/稳定性，并促进了蛋白质的表达和分泌。这些发现表明，Exin21/Qα 有可能成为一种通用的蛋白质生产增强剂，这对于生物医学研究和生物制品、药物和疫苗的开发都具有重要意义。
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