Savenkova Daria, Makarenko Irina, Nedorezova Daria, Kiyamova Ramziya, Bogdanov Mikhail
Research Laboratory "Biomarker", Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russian Federation.
Research Laboratory "Biomarker", Institute of Fundamental Medicine and Biology, Kazan Federal University, Kazan, Russian Federation; Department of Biology, University of Padua, Padua, Italy.
Anal Biochem. 2025 Aug;703:115875. doi: 10.1016/j.ab.2025.115875. Epub 2025 Apr 18.
Despite the enthusiasm and advances in the purification of native and engineered full-length membrane proteins, little attention has been paid to their fragments which could serve as attractive inspiration for function, regulation, or targeting of full-length membrane protein with therapeutic antibodies (Abs). Production of recombinant fragments of "therapeutic" membrane proteins for early-stage discovery research requires their purification to near homogeneity. It is important not only for the production of biotherapeutic antibodies but also for structural and functional studies of competitive protein-Abs, protein-protein, and lipid-protein interactions which heavily rely on the purity and quality of the isolated protein fragment of interest. The development of novel strategies for simple but still highly efficient protein purification remains a one of main research focus in the biotechnology and biomedicine because conventional purification approaches require complex manipulation steps and are timely and costly. Here, we would like to introduce a simple and rapid protein purification strategy for the human NaPi2b N-terminal (NT) sequence recombinantly expressed in a bacterial host at a laboratory scale. We demonstrate that "resin overload" e.g. the conditions when loading exceeds dynamic binding capacity can be counterintuitively but intelligently utilized to isolate highly purified protein fragments and prevent non-specific low-affinity binding of contaminant endogenous host proteins. The results showed that this method allowed us to achieve the highest purity while maintaining both immunogenic (recognition by Abs) and functional (phosphorylation) properties of the NaPi2b NT sequence. Although adaptations are required on a case-to-case basis, we believe this work can inspire other researchers working with the purification of protein and protein fragments to apply this proof-of-principle in a scalable manner.
尽管在天然和工程全长膜蛋白的纯化方面热情高涨且取得了进展，但人们对其片段的关注却很少，而这些片段可能为全长膜蛋白的功能、调节或用治疗性抗体（Abs）靶向提供有吸引力的灵感。生产用于早期发现研究的“治疗性”膜蛋白的重组片段需要将其纯化至接近均一性。这不仅对生物治疗性抗体的生产很重要，而且对竞争性蛋白-Ab、蛋白-蛋白和脂-蛋白相互作用的结构和功能研究也很重要，这些研究严重依赖于目标分离蛋白片段的纯度和质量。开发简单但仍然高效的蛋白质纯化新策略仍然是生物技术和生物医学的主要研究重点之一，因为传统的纯化方法需要复杂的操作步骤，既耗时又昂贵。在这里，我们想介绍一种简单快速的蛋白质纯化策略，用于在实验室规模下在细菌宿主中重组表达的人NaPi2b N端（NT）序列。我们证明，“树脂过载”，例如上样量超过动态结合容量的情况，可以以一种与直觉相反但明智的方式加以利用，以分离高度纯化的蛋白质片段，并防止污染性内源性宿主蛋白的非特异性低亲和力结合。结果表明，该方法使我们能够在保持NaPi2b NT序列的免疫原性（被Abs识别）和功能（磷酸化）特性的同时，实现最高纯度。尽管需要根据具体情况进行调整，但我们相信这项工作可以启发其他从事蛋白质和蛋白质片段纯化的研究人员以可扩展的方式应用这一原理验证。
