Continuous flow aqueous two phase electrophoresis -- a comparison with ultrafiltration and centrifugation

Rolle, Clinton Fabian

Student Work

Continuous flow aqueous two phase electrophoresis -- a comparison with ultrafiltration and centrifugation

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Two-phase continuous flow electrophoresis is a process currently being developed at Worcester Polytechnic Institute under the stewardship of Professor William Clark. This innovative process uses a phase system, composed of 4% Polyethylene Glycol and 6% Dextran, which selective[ly] partitions the protein into the top phase, while cell debris and other cellular components are partitioned to the bottom phase under the influence of an electric field. In addition to its ability to separate proteins from cell debris, the process also possesses the ability to partition DNA and endotoxins to the bottom phase, thereby making it a very useful process in the downstream treatment of fermentation broth. The objective of this investigation was to compare the performance of the two phase electrophoresis device to that of ultrafiltration and centrifugation. E.coli cells were grown, harvested, resuspended in deinonized water and frozen until further use. As needed, cells were lysed with lysozyme in a TRIS/BES buffer, and the resulting solution was spiked with 0.2 g/l of hemoglobin prior to processing. A factorial design was implemented for the ultrafiltration (UF) and centrifugation experiments varying concentration and crossflow rate for UF and rotor speed and time for centrifugation. Maximum recoveries of 94.7%, and 99.8% were observed for ultrafiltration, centrifugation respectively. In separate studies, electrophoretic recoveries of 99.98% were observed. High recoveries coupled with the unique ability to simultaneously remove DNA and endotoxins, clearly establishes the superiority of the process. While the cost of high purity dextran makes this process the most expensive one, if current investigations on the use of industrial grade dextran are successful, the electrophoresis process would be more efficient and economical than its competitors.

This report represents the work of one or more WPI undergraduate students submitted to the faculty as evidence of completion of a degree requirement. WPI routinely publishes these reports on its website without editorial or peer review.

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