Affinity purification of fibrinogen using an Affimer column.
Affinity purification of fibrinogen using an Affimer column.
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使用 Affimer 柱对纤维蛋白原进行亲和纯化。
DOI：
10.1016/j.bbagen.2022.130115
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Pechlivani N
中科院分区：
文献类型：
--
作者：
Pechlivani N
背景纤维蛋白原是一种含量丰富的血浆蛋白，在凝血和止血中起重要作用，因此受到广泛的研究关注。然而，蛋白质纯化是耗时的，商业制剂往往含有蛋白质污染物。本研究的目的是建立一种高质量、高功能的纤维蛋白原纯化新方法。方法用噬菌体展示系统分离纤维蛋白原特异的亲和蛋白，将其固定在SulfoLink树脂柱上，从血浆样品中纯化纤维蛋白原。纤维蛋白原用高pH溶液洗脱。商品化的人纤维蛋白原也通过亲和层析柱进一步纯化。用SDS-PAGE和质谱仪测定纤维蛋白原的纯度，用比浊法测定纤维蛋白原的功能。结果亲和分子纯化的人血浆纤维蛋白原纯度至少与市售制剂相当，并能形成生理纤维蛋白网络。用亲和层析柱进一步纯化商业上可获得的纤维蛋白原，去除了多种污染物蛋白质，其中相当一部分是凝血级联反应的关键元件，包括纤溶酶原和凝血因子XIII。结论亲和层析柱为从血浆中分离功能纤维蛋白原和进一步纯化商业上可获得的纤维蛋白原制剂提供了一种新的、快速的概念证明方法。
BackgroundFibrinogen is an abundant plasma protein with an essential role in blood coagulation and haemostasis thus receiving significant research interest. However, protein purification is time consuming and commercial preparations often have protein contaminants. The aim of this study was to develop a new method to purify high quality and functional fibrinogen.MethodsFibrinogen-specific Affimer protein, isolated using phage display systems, was immobilised to SulfoLink resin column and employed for fibrinogen purification from plasma samples. Fibrinogen was eluted using a high pH solution. Commercial human fibrinogen was also further purified using the Affimer column. Fibrinogen purity was determined by SDS-PAGE and mass spectrometry, while functionality was assessed using turbidimetric analysis.ResultsAffimer-purified fibrinogen from human plasma showed purity at least comparable to commercially available preparations and was able to form physiological fibrin networks. Further purification of commercially available fibrinogen using the Affimercolumn eliminated multiple contaminant proteins, a significant number of which are key elements of the coagulation cascade, including plasminogen and factor XIII.ConclusionsThe Affimercolumn represents a proof of concept novel, rapid method for isolating functional fibrinogen from plasma and for further purification of commercially available fibrinogen preparations.General significanceOur methodology provides an efficient way of purifying functional fibrinogen with superior purity without the need of expensive pieces of equipment or the use of harsh conditions.
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