EXPRESSION AND PURIFICATION OF A SOLUBLE RECOMBINANT A1 DOMAIN OF HUMAN VON WILLEBRAND FACTOR IN BACTERIA

Von Willebrand factor (VWF) is a multimeric, plasma glycoprotein that plays an essential role in hemostasis and thrombosis. The A1 domain of VWF contains multiple binding sites with critical roles in the initiation of platelet aggregation. The relationship of structure to function in the VWF A1 domain is a topic of intense interest, both from the perspective of understanding how the function of this domain is regulated in vivo and from the standpoint of developing novel antithrombotic agents. An isolated A1 domain expressed in recombinant form can serve as an invaluable tool for studying its structural and functional attributes. However, problems with solubility have frequently been encountered in generating a recombinant form of the A1 domain. Bacterial expression, in particular, has invariably resulted in accumulation of the recombinant protein in inclusion bodies. We have generated a soluble form of the recombinant A1 domain of VWF in bacteria through a vector with a C-terminal hexahistidine tag. This method permits extraction and purification of the recombinant A1 domain protein entirely under native conditions, thereby preserving its non-covalent and disulfide bonds.