摘要：In order to initiate the formation of a platelet plug Von Willebrand Factor (VWF) must
be assembled into large multimers. VWF undergoes post translational modifications
by dimerizing through multiple intermolecular disulfide bonds between carboxyl
terminal ends of the protein and once in Golgi by forming interdimer disulfide bonds.
The resulting multimers range in size between 500 to 20000 kDa. The protein
dimerizes and the dimers then form a variety of disulfide crosslinked multimers with
as many as 80 monomeric units, weighing more than 20 million Daltons. Studying
such an enormous molecule poses special challenges. The separate domains within the
VWF subunit exhibit specific properties, involving interactions with other molecules.
Binding sites that are independent of multimer assembly but important for the
hemostatic function are located in the A1A2A3 domains of VWF. We expressed the
A1, A2, and A3 domains of von Willebrand factor in a single polypeptide using Pichia
pastoris expression system. Proteins with disulfide bonds, requiring post translational
modifications and glycosylation can be produced in their correctly native folded states
with full function from Pichia pastoris. We purified the A1A2A3 domain using
ethanol, ammonium sulfate precipitation and ion exchange chromatography. Our
efforts in solubilizing the purified protein were unsuccessful more likely due to the
unusual adhesive nature of the A1A2A3 domain of the VWF.
关键词：Von Willerband Factor; VWF A1A2A3 Domains; Pichia pastoris;
Protein Purification
