Heterologous glycoprotein production relies on host glycosylation-dependent folding. When the biosynthetic machinery differs from the usual expression host, there is scope to remodel the assembly pathway to enhance glycoprotein production. Here we explore the integration of chaperone coexpression with glyco-engineering to improve the production of a model HIV-1 envelope antigen. Calreticulin was coexpressed to support protein folding together with Leishmania major STT3D oligosaccharyltransferase, to improve glycan occupancy, RNA interference to suppress the formation of truncated glycans, and Nicotiana benthamiana plants lacking α1,3-fucosyltransferase and β1,2-xylosyltransferase was used as an expression host to prevent plant-specific complex N-glycans forming. This approach reduced the formation of undesired aggregates, which predominated in the absence of glyco-engineering. The resulting antigen also exhibited increased glycan occupancy, albeit to a slightly lower level than the equivalent mammalian cell-produced protein. The antigen was decorated almost exclusively with oligomannose glycans, which were less processed compared with the mammalian protein. Immunized rabbits developed comparable immune responses to the plant-produced and mammalian cell-derived antigens, including the induction of autologous neutralizing antibodies when the proteins were used to boost DNA and modified vaccinia Ankara virus-vectored vaccines. This study demonstrates that engineering glycosylation-directed folding offers a promising route to enhance the production of complex viral glycoproteins in plants.

中文翻译：

---

增强糖基化导向的折叠途径可增强植物中 HIV Env 免疫原产生的保真度

异源糖蛋白的产生依赖于宿主糖基化依赖性折叠。当生物合成机制与通常的表达宿主不同时，就有可能重塑组装途径以提高糖蛋白的产量。在这里，我们探索伴侣共表达与糖工程的整合，以改善模型 HIV-1 包膜抗原的生产。钙网蛋白与利什曼原虫主要STT3D寡糖基转移酶一起共表达以支持蛋白质折叠，以提高聚糖占有率，RNA干扰以抑制截短聚糖的形成，并且使用缺乏α1,3-岩藻糖基转移酶和β1,2-木糖基转移酶的本塞姆氏烟草植物作为表达宿主以防止植物特有的复合N-聚糖形成。这种方法减少了不需要的聚集体的形成，这种聚集体在没有糖工程的情况下占主导地位。所得抗原还表现出聚糖占有率增加，尽管其水平略低于同等的哺乳动物细胞产生的蛋白质。该抗原几乎完全由寡甘露糖聚糖修饰，与哺乳动物蛋白质相比，其加工较少。免疫兔子对植物产生的抗原和哺乳动物细胞衍生的抗原产生了类似的免疫反应，包括当使用蛋白质增强 DNA 和修饰的安卡拉痘苗病毒载体疫苗时诱导自体中和抗体。这项研究表明，糖基化定向折叠工程为增强植物中复杂病毒糖蛋白的生产提供了一条有前途的途径。