Nanobodies are single-domain antibody constructs derived from the variable regions of heavy chain only (V_HH) 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 Ni²⁺ 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.

纳米抗体是源自仅重链 (V _H H) 骆驼 IgG 可变区的单域抗体构建体。它们的小尺寸和单基因形式使它们适用于需要基于蛋白质亲和力的方法的各种分子生物学应用。这些特征，除了它们的高溶解度外，还允许它们在大肠杆菌系统中的周质表达、提取和纯化，使用标准化的协议相对容易。然而，一些纳米抗体对大肠杆菌内的周质表达、提取和纯化具有抗拒性系统。为了改善它们的表达，需要改变表达宿主、载体或增加表达规模，所有这些都需要增加表达的复杂性和生产成本。然而，如此处所示，现有标准大肠杆菌培养方案中的具体变化旨在减少选择性抗生素压力的破坏、增加初始培养接种量和改善向周质空间的运输，从而挽救了几种此类耐火纳米抗体的表达。周质提取方案也进行了更改，以确保有效的渗透作用，防止蛋白质降解并防止下游的 Ni ^2+螯合IMAC 纯化过程中的离子。采用该协议将总体上改善纳米抗体的表达，特别是那些顽固的纳米抗体。