Production and compositional analysis of full-length influenza virus hemagglutinin in Nanodiscs: Insights from multi-angle light scattering

IF 1.4 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS Protein expression and purification Pub Date : 2024-12-07 DOI:10.1016/j.pep.2024.106641

Tim G.J. Knetsch, Marcellus Ubbink

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Abstract

The global threat of pandemics highlights the urgency of developing innovative vaccine strategies. Viral spike proteins are the primary antigens recognized by the immune system and serve as key targets for vaccine development. This study reports the production of full-length Influenza A virus surface glycoprotein, hemagglutinin (HA), and its incorporation into Nanodiscs (NDs). HA was expressed in insect cells and purified using detergents, maintaining its functional integrity. Characterisation by size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) confirmed that HA could be incorporated into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) NDs as a single trimer. SEC-MALS was instrumental in analysing the composition of NDs, which included HA, membrane scaffold proteins, lipids, and glycans. These findings provide a robust framework for the production and reconstitution of glycoproteins in NDs, and offers valuable insights into the study of multi-component nanoparticles using MALS. Our work highlights the potential of NDs for studying viral glycoproteins and advances the development of well-defined recombinant ND-based vaccines.

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纳米盘中全长流感病毒血凝素的生产和成分分析：多角度光散射的启示。

流行病的全球威胁凸显了制定创新疫苗战略的紧迫性。病毒刺突蛋白是免疫系统识别的主要抗原，是疫苗开发的关键靶点。本研究报道了全长甲型流感病毒表面糖蛋白血凝素（HA）的产生及其与纳米片（NDs）的结合。HA在昆虫细胞中表达，并用洗涤剂纯化，保持其功能完整性。通过尺寸排除色谱和多角度光散射（SEC-MALS）表征，证实HA可以作为一个三聚体并入1-棕榈酰-2-油基-sn-甘油-3-磷脂胆碱（POPC） ndds中。SEC-MALS有助于分析NDs的组成，其中包括HA、膜支架蛋白、脂质和聚糖。这些发现为NDs中糖蛋白的生成和重组提供了一个强有力的框架，并为利用MALS研究多组分纳米颗粒提供了有价值的见解。我们的工作强调了NDs在研究病毒糖蛋白方面的潜力，并推进了明确定义的重组NDs疫苗的开发。

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来源期刊

Protein expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

期刊介绍： Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.