Production of extracellular domain of human tissue factor using maltose-binding protein fusion system.
Making use of the physiological process of coagulation as an anti-tumor effector function may be beneficial in various coagulation-mediated diseases. Preclinical and clinical studies with novel tissue factor targeting constructs require that efficient procedures for preparing large quantities of pure truncated TF (tTF) become available. In this study, we described a simple and rapid on-column method for purifying large quantities of human tTF from Escherichia coli. The coding region of extracellular domain of tissue factor was linked to the 3(')-end of maltose-binding protein (MBP) gene. The fusion protein was expressed as soluble form after induction by isopropylthio-beta-D-galactoside (IPTG). MBP-tTF was purified by amylose affinity chromatography. MBP can be removed by digestion with factor Xa. Expression could represent 21.5% of the total soluble protein in E. coli, allowing approximately 15mg of highly purified protein to be obtained per liter of bacterial culture. The fusion protein was recognized in Western blot by anti-TF monoclonal antibody and the activity was confirmed by chromogenic assay. This MBP-fusion system permits large-scale functional expression and purification of recombinant soluble proteins, providing a basis for the future study of structure and function of tTF.
Guan
Ming
M
Department of Laboratory Medicine, Hua Shan Hospital, Fudan University, 12 Central Urumqi Road, 200040, Shanghai, People's Republic of China.
Su
Bing
B
Ye
Chuanzhong
C
Lu
Yuan
Y
D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013925:Thromboplastin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D013925:Thromboplastin