Date of Award

Summer 1998

Document Type

Thesis

Degree Name

Master of Science in Chemical Engineering - (M.S.)

Department

Chemical Engineering, Chemistry and Environmental Science

First Advisor

Kamalesh K. Sirkar

Second Advisor

Robert G. Luo

Third Advisor

Norman W. Loney

Abstract

Electroporation is commonly used to transfer genetic materials such as DNA into the cell by exposing the cell to a high electric field that momentarily permeabilizes the cell membrane. This premise was utilized to use electroporation as a means of releasing intracellular proteins. A procedure has been developed for the release of intracellular proteins through the use of electroporation. This procedure is a breakthrough. In general, industrial scale techniques to recover proteins from microorganisms are inefficient (Clarkson, 1993). A benefit of this electroporation-based mechanism would be the reduction of steps in the downstream processing required to purify the desired product.

Yeast cells in suspensions were subjected to high voltage electric pulses by using an E.coli Pulser from BIO-RAD. The pulses permeabilized the cell and allowed the release of intracellular materials. Research has shown that the amount of released materials increased with the number of times the cells were subjected to pulses. In addition, experiments show that even more material can be released by repulsing the cells. Other parameters to affect the release of intracellular materials were optical density, growth stage, and the storage of the cell suspensions. The protein assay was found to be an effective means to determine the released intracellular product concentration.

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