Facultad de Ciencias Exactas y Naturales, Departamento de Fisiología y Biología Molecular y Celular, Instituto de Biociencias, Biotecnología y Biología Traslacional (iB3), Universidad de Buenos Aires, Intendente Güiraldes 2160, C1428EGA, Buenos Aires, Argentina.
Facultad de Farmacia y Bioquímica, Departamento de Química Biológica, Universidad de Buenos Aires, Junín 965 C1113AAD. Buenos Aires, Argentina.
Glycobiology. 2024 Mar 19;34(1). doi: 10.1093/glycob/cwad089.
During the COVID-19 outbreak, numerous tools including protein-based vaccines have been developed. The methylotrophic yeast Pichia pastoris (synonymous to Komagataella phaffii) is an eukaryotic cost-effective and scalable system for recombinant protein production, with the advantages of an efficient secretion system and the protein folding assistance of the secretory pathway of eukaryotic cells. In a previous work, we compared the expression of SARS-CoV-2 Spike Receptor Binding Domain in P. pastoris with that in human cells. Although the size and glycosylation pattern was different between them, their protein structural and conformational features were indistinguishable. Nevertheless, since high mannose glycan extensions in proteins expressed by yeast may be the cause of a nonspecific immune recognition, we deglycosylated RBD in native conditions. This resulted in a highly pure, homogenous, properly folded and monomeric stable protein. This was confirmed by circular dichroism and tryptophan fluorescence spectra and by SEC-HPLC, which were similar to those of RBD proteins produced in yeast or human cells. Deglycosylated RBD was obtained at high yields in a single step, and it was efficient in distinguishing between SARS-CoV-2-negative and positive sera from patients. Moreover, when the deglycosylated variant was used as an immunogen, it elicited a humoral immune response ten times greater than the glycosylated form, producing antibodies with enhanced neutralizing power and eliciting a more robust cellular response. The proposed approach may be used to produce at a low cost, many antigens that require glycosylation to fold and express, but do not require glycans for recognition purposes.
在 COVID-19 疫情期间，已经开发出了许多工具，包括基于蛋白质的疫苗。甲醇营养型酵母巴斯德毕赤酵母（同义词为 Komagataella phaffii）是一种真核生物，具有成本效益高和可扩展的重组蛋白生产系统，具有高效分泌系统和真核细胞分泌途径的蛋白质折叠辅助作用。在之前的工作中，我们比较了 SARS-CoV-2 刺突受体结合域在巴斯德毕赤酵母中的表达与在人类细胞中的表达。尽管它们的大小和糖基化模式不同，但它们的蛋白质结构和构象特征是不可区分的。然而，由于酵母表达的蛋白质中的高甘露糖聚糖延伸可能是引起非特异性免疫识别的原因，我们在天然条件下对 RBD 进行了去糖基化。这导致了一种高度纯、同质、正确折叠和单体稳定的蛋白质。这通过圆二色性和色氨酸荧光光谱以及 SEC-HPLC 得到了证实，这些光谱与在酵母或人类细胞中产生的 RBD 蛋白相似。去糖基化的 RBD 可以在一步中以高产率获得，并且能够有效地区分来自患者的 SARS-CoV-2 阴性和阳性血清。此外，当使用去糖基化变体作为免疫原时，它会引起比糖基化形式强十倍的体液免疫反应，产生具有增强中和能力的抗体，并引发更强烈的细胞反应。所提出的方法可用于以低成本生产许多需要糖基化折叠和表达但不需要糖基化用于识别目的的抗原。
