Date of Award

5-6-2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

George Pierce

Second Advisor

Sidney Crow

Third Advisor

Eric Gilbert

Abstract

The ideal vaccine can generate a strong immune reaction without adverse effects on the body. Thus, many vaccines are now created using recombinant technology to accomplish this goal. Purification of recombinant proteins produced in Gram negative bacteria (GNB) presents several challenges, including reducing the concentration of contaminating host cell molecules to nontoxic levels. The most prevalent host cell molecule is lipopolysaccharide (LPS), which is a major constituent of the outer membrane of GNB. Residual LPS in final product presents a major problem for proteins intended for pharmaceutical application, as it is toxic to mammalian cells. An orthogonal approach is an FDA requirement for preparation of proteins intended for use as pharmaceuticals. The ideal approach not only reduces contaminants to acceptable measures, but also results in a high yield of properly folded protein, while maintaining an expeditious time table and keeping costs low. Though a truly universal scheme for processing proteins from GNB is not possible, a comprehensive study of scalable and certifiable methods currently used for protein purification will be performed on multiple constructs in order to outline general principles for the system in a helpful blueprint, evaluating the effectiveness of different methods of retrieval of protein from inclusion bodies in particular. This approach is based upon the hypothesis that production of fusion proteins in the insoluble fraction results in a greater yield of pure protein with fewer processing steps.

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