Date of Award

1998

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Abstract

Heterologous proteins are proteins produced by foreign hosts. The ability to overexpress commercially important proteins in various biological systems has revolutionized the field of biotechnology. In this project three proteins, namely Taq DNA polymerase, E. coli β-galactosidase, and S. cerevisiae topoisomerase I, were cloned and overexpressed in heterologous host. Conventional methods to obtain DNA polymerase from T. aquaticus were cumbersome. Cloning of the gene had been reported but the purification scheme required several steps. To simplify the purification procedure and improve the yield, the DNA polymerase gene from T. aquaticus was cloned into E. coli expression vector pUC18. A single purification step involving heat treatment resulted in approximately 8 million units of homogeneous, research-grade enzyme with aspecific activity of 3.4 × 10 6 . The yields were two orders of magnitude higher when compared to the previously available protocols. The bacterial lacZ gene exhibits intracistronic complementation, which is the basis of the α-complementation of β-galactosidase, in E. coli . In this project, an α-complementation system was established in the yeast S. cerevisiae . This was accomplished by constructing two yeast expression vectors, one containing the bacterial α fragment and another containing the ω fragment. The vectors when co-transformed into yeast produced functional β-galactosidase, and resulted in blue colonies on an X-Gal plate. This project generated a potential tool for some yeast cloning experiments and in vivo transcription studies. Eukaryotic topoisomerases are of importance in biological research. To generate an inexpensive source of topoisomerase I for in vitro transcription studies in our laboratory, the gene from the yeast S. cerevisiae was cloned and overexpressed in E. coli . At least one million units of active enzyme were obtained from 100 ml culture. The purification of this protein is currently in progress. This research demonstrates successful cloning and expression of heterologous proteins in various systems and exemplifies the power of biotechnology.

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