Expression and Purification of Recombinant Von Willebrand Factor A1A2A3 Domains
Journal Title: Journal of BioScience and Biotechnology - Year 2017, Vol 6, Issue 2
Abstract
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.
Authors and Affiliations
Esra Seyran, Rohana Liyanage, Jackson Lay
Keywords
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