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.
Highlights
IntroductionHuman fibrinogen (fgn) is a soluble 340 kDa multimeric glycoprotein found in blood plasma
Published: 19 January 2022Human fibrinogen is a soluble 340 kDa multimeric glycoprotein found in blood plasma
Control wells with only transfection reagents (PEI and ExpiFectamineTM 293) showed comparable cell viability values (≥91% and ≥85%, respectively) for the first four days, while wells containing only cells displayed higher cell viability values (≥95% and ≥94%, respectively) for the first four days. Despite their similarities in cell viabilities, cells transfected with the 3P system using PEI provided higher live cell densities over the five days post-transfection than cells transfected with the 3P system using ExpiFectamineTM 293
Summary
Human fibrinogen (fgn) is a soluble 340 kDa multimeric glycoprotein found in blood plasma. The normal range of circulating fibrinogen levels varies slightly based on gender, age, geographic region, race, and ethnicity, and can vary slightly among hematology laboratories depending on the measurement techniques utilized [1]. Fibrinogen, and its cleavage product fibrin, participate in many important biological functions including hemostasis, angiogenesis, wound healing, and inflammation among others [3,4,5,6]. Fibrin’s action in these processes is enabled by its remarkable mechanical properties, including extensibility and elasticity rivaling rubber [7,8,9,10]. Fibrin-based sealants have been used for tissue engineering, tissue regeneration, stem cell and platelet delivery, and angiogenesis [16].
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