Affinity purification of fibrinogen using an Affimer column

• Published in: Biochimica et biophysica acta. General subjects Vol. 1866; no. 5; p. 130115
• Main Authors: Pechlivani, Nikoletta, Kearney, Katherine J., Tiede, Christian, Cheah, Ramsah, Phoenix, Fladia, Ponnambalam, Sreenivasan, Ault, James R., McPherson, Michael J., Tomlinson, Darren C., Ajjan, Ramzi A.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.05.2022
• Subjects: Affimer; Affinity column; bacteriophages; blood coagulation; Chromatography, Affinity - methods; coagulation; fibrin; Fibrin - metabolism; fibrinogen; Fibrinogen - metabolism; Fibrinogen purification; Hemostasis; Humans; mass spectrometry; Plasma; Plasminogen; polyacrylamide gel electrophoresis; rapid methods; Affimer; Plasma; Affinity column; Fibrinogen purification
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2022.130115

Fibrinogen 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. Fibrinogen-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. Affimer-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. The Affimercolumn represents a proof of concept novel, rapid method for isolating functional fibrinogen from plasma and for further purification of commercially available fibrinogen preparations. Our 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. •Proof of concept novel methodology employs fibrinogen-specific Affimer protein for fibrinogen purification from plasma.•Rapid method for further purification of commercially available fibrinogen•Purified fibrinogen has superior purity and retains its biological function.