Laboratory of Cellular and Molecular Interactions (CMIM), Vrije Universiteit Brussels, Brussels, Belgium; Department of Tropical and Infectious Diseases, Institute of Primate Research (IPR), Nairobi, Kenya.
Laboratory of Cellular and Molecular Interactions (CMIM), Vrije Universiteit Brussels, Brussels, Belgium; Laboratory for Biomedical Research, Ghent University Global Campus, Yeonsu-Gu, Incheon, South Korea; Department of Biochemistry and Microbiology, Universiteit Gent, Ledeganckstraat 35, 9000, Gent, Belgium.
Protein Expr Purif. 2021 Sep;185:105906. doi: 10.1016/j.pep.2021.105906. Epub 2021 May 12.
Nanobodies are single-domain antibody constructs derived from the variable regions of heavy chain only (VH) camelid IgGs. Their small size and single gene format make them amenable to various molecular biology applications that require a protein affinity-based approach. These features, in addition to their high solubility, allows their periplasmic expression, extraction and purification in E. coli systems with relative ease, using standardized protocols. However, some Nanobodies are recalcitrant to periplasmic expression, extraction and purification within E. coli systems. To improve their expression would require either a change in the expression host, vector or an increased scale of expression, all of which entail an increase in the complexity of their expression, and production cost. However, as shown here, specific changes in the existing standard E. coli culture protocol, aimed at reducing breakdown of selective antibiotic pressure, increasing the initial culture inoculum and improving transport to the periplasmic space, rescued the expression of several such refractory Nanobodies. The periplasmic extraction protocol was also changed to ensure efficient osmolysis, prevent both protein degradation and prevent downstream chelation of Ni ions during IMAC purification. Adoption of this protocol will lead to an improvement of the expression of Nanobodies in general, and specifically, those that are recalcitrant.
纳米抗体是源自重链可变区（VH）骆驼 IgG 的单域抗体构建体。它们的体积小且基因单一，适用于需要基于蛋白质亲和力的各种分子生物学应用。除了高溶解性外，这些特性还允许它们在大肠杆菌系统中以相对简单的方式进行周质表达、提取和纯化，使用标准化的方案。然而，一些纳米抗体在大肠杆菌系统中周质表达、提取和纯化方面存在困难。为了提高它们的表达水平，要么需要改变表达宿主、载体，要么需要增加表达规模，所有这些都需要增加它们表达的复杂性和生产成本。然而，如这里所示，对现有标准大肠杆菌培养方案进行特定的改变，旨在减少选择抗生素压力的崩溃，增加初始培养接种物，并改善向周质空间的转运，可以挽救几种此类难表达的纳米抗体的表达。周质提取方案也进行了更改，以确保有效渗透，防止蛋白质降解并防止 IMAC 纯化过程中 Ni 离子的下游螯合。采用该方案将普遍提高纳米抗体的表达水平，特别是那些难以表达的纳米抗体。
