Development of Transient Recombinant Expression and Affinity Chromatography Systems for Human Fibrinogen

Abstract

Fibrin forms the structural scaffold of blood clots and has great potential for biomaterial applications. Creating recombinant expression systems of fibrinogen, fibrin’s soluble precursor, would advance the ability to construct mutational libraries that would enable structure–function studies of fibrinogen and expand the utility of fibrin as a biomaterial. Despite these needs, recombinant fibrinogen expression systems, thus far, have relied on the time-consuming creation of stable cell lines. Here we present tests of a transient fibrinogen expression system that can rapidly generate yields of 8–12 mg/L using suspension HEK Expi293TM cells. We report results from two different plasmid systems encoding the fibrinogen cDNAs and two different transfection reagents. In addition, we describe a novel, affinity-based approach to purifying fibrinogen from complex media such as human plasma. We show that using a high-affinity peptide which mimics fibrin’s knob ‘A’ sequence enables the purification of 50–75% of fibrinogen present in plasma. Having robust expression and purification systems of fibrinogen will enable future studies of basic fibrin(ogen) biology, while paving the way for the ubiquitous use of fibrin as a biomaterial.

Potential Application of Recombinant Snake Prothrombin Activator Ecarin in Blood Diagnostics

“…In E. coli expression systems, it has been estimated that modest protein expression of recombinant protein is between 50 and 150 mg/L (2–5% of the total cellular protein) and soluble proteins can be recovered with good yields (>50%) [ 39 ]. Recently, transient fibrinogen expression has been shown to rapidly generate yields of 8–12 mg/L fibrinogen in HEK (Expi293 TM ) suspension cells, with purification recovery of 50–75% of the recombinant fibrinogen [ 40 ]. According to the Jonebring et al study, the highest yield of CHO- produced ecarin was up to 7000 EU ecarin/litre in lab scale shaker cultures (equivalent to ~5500 mU/L), but the recovery percentage of the purified rEcarin was not reported [ 13 ].…”

Recent Advances Regarding Polyphenol Oxidase in Camellia sinensis: Extraction, Purification, Characterization, and Application

“…Affinity chromatography is a protein purification method, which is designed based on the specific and reversible binding between proteins and matrices [43]. Nickel column affinity chromatography is a widely used method for purifying recombinant proteins [67]. Due to the competitive binding of Ni 2+ ions in nickel columns to imidazole or proteins with His-Tag, increasing the concentration of imidazole in the elution buffer can elute the target protein to achieve protein purification.…”

Affinity purification of fibrinogen using an Affimer column

“…Other methods include cryo-, ammonium sulfate or β-alanine precipitation, chromatography methods using ristocetin agarose, protamine-agarose or n-alkylagaroses [6,[9][10][11][12][13][14][15]. Affinity of Staphylococcus aureus clumping factor A [7] and synthetic peptides have also been used for fibrinogen purification [16][17][18] as well as different monoclonal antibodies [19,20]. Although all the methodologies yield purified protein, there are drawbacks that should not be underestimated.…”