Abstract Cyanovirin-N (CV-N), a lectin of cyanobacterial origin, has important medical implications due to its highly effective virucidal activity. However, its low production yield has limited its application till date. In this study, we demonstrate for the first time, a simple process for a break-through production of soluble and bioactive recombinant CV-N (rCV-N) in a newer strain called Escherichia coli SHuffle® T7 Express lysY that is engineered for enhanced production of correctly disulphide bonded proteins. As rCV-N contains two critical disulphide bonds required for its anti-HIV activity, rational choice of this expression host could produce high yield of soluble rCV-N (∼24 mg/ 100 ml) in simple Erlenmeyer flask at 20 °C. The protein could be obtained to near purity in single-step affinity purification (mixture of monomer, dimer and higher order oligomers together referred to as mixed form) which was further resolved into monomer and dimer by size exclusion chromatography. The purified rCV-N had a monomeric mass of 11.962 kDa and a prominent β-sheet secondary structure. The three forms of rCV-N i.e. mixed, monomer and dimer exhibited significant anti-HIV activity (IC50 0.5−5 nM) and a therapeutic index of ∼1,000–10,000 in vitro (negligible cytotoxicity up to 5 μM). All rCV-N forms had low endotoxins.

中文翻译：

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从合理选择的大肠杆菌菌株中高产地生产具有显着抗 HIV 活性的重组蓝病毒素-N（抗病毒凝集素）

摘要 Cyanovirin-N (CV-N) 是一种源自蓝藻的凝集素，由于其高效的杀病毒活性而具有重要的医学意义。然而，其低产量限制了其应用至今。在这项研究中，我们首次展示了在一种名为大肠杆菌 SHuffle® T7 Express lysY 的新型菌株中突破性生产可溶性和生物活性重组 CV-N (rCV-N) 的简单过程，该菌株旨在增强生产正确的二硫键蛋白。由于 rCV-N 包含其抗 HIV 活性所需的两个关键二硫键，因此合理选择该表达宿主可以在 20°C 的简单锥形瓶中产生高产率的可溶性 rCV-N（~24 mg/100 ml）。通过一步亲和纯化（单体、二聚体和更高级的低聚物统称为混合形式），通过尺寸排阻色谱将其进一步拆分为单体和二聚体。纯化的 rCV-N 具有 11.962 kDa 的单体质量和突出的 β-折叠二级结构。rCV-N 的三种形式，即混合型、单体型和二聚体，表现出显着的抗 HIV 活性（IC50 0.5-5 nM）和体外治疗指数约为 1,000-10,000（高达 5 μM 的细胞毒性可忽略不计）。所有 rCV-N 形式都具有低内毒素。5-5 nM) 和 ∼1,000–10,000 体外 治疗指数（高达 5 μM 的细胞毒性可忽略不计）。所有 rCV-N 形式都具有低内毒素。5-5 nM) 和 ∼1,000–10,000 体外 治疗指数（高达 5 μM 的细胞毒性可忽略不计）。所有 rCV-N 形式都具有低内毒素。