Designing Protein Expression and Purification Systems for Recombinant Z Alpha1-Antitrypsin using the Methylotrophic Yeast, Pichia pastoris

Author

Monique Jenaie LemieuxFollow

Date of Award

8-2012

Degree Type

Thesis

Degree Name

Master of Science

Major

Life Sciences

Major Professor

Valerie Berthelier

Committee Members

N. Jain, H. O'Neill, J. Baudry

Abstract

It is well established that improper protein folding often leads to the formation of aggregates whose consequences are cellular impairment and cell death. One example of this is the aggregation of the mutant Z Alpha1-Antitrypsin protein, which results in blocking of its secretion due to inclusion body formation. This can contribute not only to the development of chronic obstructive pulmonary disease but also to hepatitis, cirrhosis and hepatocellular carcinoma. Current treatments are principally limited to intravenous Alpha1-Antitrypsin therapy and organ transplantation. In the scientific community though, it is widely thought that more effective forms of treatments lie within the polymerization process itself. However, in order to conduct the necessary experiments for this, copious amounts of Alpha1-Antitrypsin protein are needed. The amounts far out-weigh those that are obtainable through traditional methods of plasma purification. Therefore there is a high demand for methods that can successfully produce recombinant Alpha1-Antitrypsin protein. Over the years, several systems have been implemented to produce wild-type Alpha1-Antitrypsin protein but it was not until 2009 that recombinant Z Alpha1-Antitrypsin protein was successfully produced. The goal of this thesis was to expand upon this original study in order to produce both untagged and tagged versions of recombinant Z Alpha1-Antitrypsin protein using the methylotrophic yeast, Pichia pastoris. Colonies from the protease-deficient strand SMD1163 were selected for multi-copy integrations and then purified using affinity chromatography and anion exchange chromatography. While the results of the purification process proved inconclusive, they do help lay the groundwork for future endeavors.

Recommended Citation

Lemieux, Monique Jenaie, "Designing Protein Expression and Purification Systems for Recombinant Z Alpha1-Antitrypsin using the Methylotrophic Yeast, Pichia pastoris. " Master's Thesis, University of Tennessee, 2012.
https://trace.tennessee.edu/utk_gradthes/1317