Voitechovič Edita, Korepanov Anton, Kirsanov Dmitry, Legin Andrey
St. Petersburg State University, St. Petersburg, Russia; Institute of Microelectronics of Barcelona, Barcelona, Spain.
Protein Contour LLC, St. Petersburg, Russia.
J Pharm Biomed Anal. 2018 Feb 20;150:67-71. doi: 10.1016/j.jpba.2017.11.076. Epub 2017 Dec 5.
Quantification of proteins is a key biochemical assay in molecular biology, biotechnology, medicine and pharmacology. Protein quantification protocols can be based on spectrophotometry, enzyme-linked immunosorbent assay, mass spectrometry or quantitative immunoblotting depending on analyte. In case of immobilized protein these methods require suitable sample preparation. Thus, sophisticated analysis becomes even more complex, expensive and time-consuming. Such drawbacks are highly undesirable in industry. In this study we propose a new approach for evaluation of immobilized protein concentration based on application of bio-assisted potentiometric multisensor system. Surface-immobilized recombinant protein A from Staphylococcus aureus (SpA, expressed in Escherichia coli), which is commonly used as affinity ligand immobilized to stationary phase (сhromatography media) for monoclonal antibody purification was employed as the model object. Chromatography media samples containing different amounts of immobilized SpA were analyzed. Proteinase K from Tritirachium album was employed as a bio-transducer. We demonstrated that the suggested approach provides information about immobilized SpA concentration with 0.8mg/ml accuracy in the range 1-6.7mg/ml and within just 16min. Moreover, the proposed procedure requires no expensive materials and equipment and no bio-transducer immobilization. This method has potential of application for fast monitoring of other immobilized proteins in different tasks.
蛋白质定量是分子生物学、生物技术、医学和药理学中的一项关键生化分析方法。蛋白质定量方案可基于分光光度法、酶联免疫吸附测定、质谱分析或定量免疫印迹法，具体取决于分析物。对于固定化蛋白质，这些方法需要合适的样品制备。因此，复杂的分析变得更加复杂、昂贵且耗时。这些缺点在工业中是非常不可取的。在本研究中，我们提出了一种基于生物辅助电位多传感器系统应用来评估固定化蛋白质浓度的新方法。以表面固定化的金黄色葡萄球菌重组蛋白A（SpA，在大肠杆菌中表达）为模型对象，该蛋白通常用作固定在固定相（色谱介质）上的亲和配体，用于单克隆抗体的纯化。对含有不同量固定化SpA的色谱介质样品进行了分析。选用来自米曲霉的蛋白酶K作为生物传感器。我们证明，所建议的方法能够在1 - 6.7mg/ml范围内以0.8mg/ml的准确度提供有关固定化SpA浓度的信息，且仅需16分钟。此外，所提出的方法不需要昂贵的材料和设备，也无需固定生物传感器。该方法具有在不同任务中快速监测其他固定化蛋白质的应用潜力。
