A new feasible method for fibrinogen purification based on the affinity of Staphylococcus aureus clumping factor A to fibrinogen.
Plasma fibrinogen participates in several physiological and pathological events thus becoming a useful studying material in biomedical research. Here we report a new convenient method for fibrinogen purification based on the affinity of Staphylococcus aureus clumping factor A to fibrinogen. Clumping factor A (ClfA) is a cell wall-anchored surface protein of S. aureus bacteria that binds with a high affinity to the fibrinogen gamma chain C-terminus via a segment encompassing the residues 221-550. This activity of ClfA (ClfA(221-550)) was produced in fusion to the C-terminus of glutathione-S-transferase (GST) with recombinant technology and used as an affinity ligand to capture plasma fibrinogen. GST-ClfA(221-550) fusion protein was immobilized onto the glutathione-conjugated beads packed in a plastic column by its GST part. Then, this affinity column was loaded with citrated and heparinized human plasma. After washing out unbound proteins, column-captured fibrinogen was specifically eluted down with a citrate buffer solution (50mM, pH 5.6). Purified human fibrinogen exhibited the ability to support platelet adhesion and aggregation and formed fibrin clot by thrombin, indicating that ClfA(221-550)-purified human fibrinogen is a functionally active product. We also found that both the rat and mouse fibrinogens could be purified as well as human fibrinogen with this method. By virtue of its simplicity and feasibility, ClfA(221-550)-based method would be very useful to the investigators who need fibrinogen to perform their studies.
Liu
Chao-Zong
CZ
Department of Pharmacology, College of Medicine, Tzu Chi University, No. 701, Chung-Yung Road, Sec. 3, Hualien City, Hualien County, Taiwan. czliu33@mail.tcu.edu.tw
Cheng
Hui-Ju
HJ
Chang
Ling-Ya
LY
D000818:Animals; D001483:Base Sequence; D002448:Cell Adhesion; D002846:Chromatography, Affinity; D003030:Coagulase / Q000378:metabolism*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005240:Feasibility Studies; D005340:Fibrinogen / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D051379:Mice; D010974:Platelet Aggregation; D051381:Rats
C515944:ClfA protein, Staphylococcus aureus; D003030:Coagulase; D017931:DNA Primers; D005340:Fibrinogen