Date of Award

5-2013

Document Type

Thesis

Degree Name

Master of Science (MS)

Legacy Department

Bioengineering

Advisor

Harcum, Sarah W

Committee Member

Alexis , Frank

Committee Member

Sehorn , Michael G

Abstract

Escherichia coli is used intensively for recombinant protein production due to its many unique advantages, but one key challenge with the use of E. coli is the tendency of recombinant proteins to misfold and aggregate into insoluble inclusion bodies (IBs). The presence of IBs stresses cells and can hinder overall growth. Additionally, IBs contain high concentrations of recombinant protein in an inactive form and thus require recovery steps to salvage functional recombinant protein. Currently, no universally effective method exists to prevent IB formation in recombinant E. coli. Further research into the gene expression response to insoluble recombinant protein may provide insight into critical cellular mechanisms that could be leveraged to minimize IB formation. This study was focused on characterizing the dynamic transcriptional response of E. coli in the initial stages of IB formation, as previous studies have only characterized gene expression changes after IBs had accumulated.
In this study, DNA microarrays were used to compare the E. coli gene expression response to soluble and to insoluble recombinant protein production. Genes involved in many several cellular functionalities were differentially expressed due to the production of insoluble recombinant protein. As expected and previously reported, expression levels of many classical heat-shock genes increased, including protein folding chaperones and proteases. Additionally, cells increased expression levels of protein synthesis-related genes and of genes involved in energy-deriving pathways. Interestingly, expression levels decreased for many transmembrane transporter genes for many substances not found in the culture medium, while several genes involved in catabolic pathways for these substances also decreased in expression. Additionally, over a third of the differentially expressed genes were classified as putative genes, indicating that IB stress regulates many genes that have not been extensively studied. Taken together, the results of this study indicate that IB formation in recombinant E. coli is a complex issue that not only induces the heat-shock genes but also directly causes the cells to increase protein and energy synthesis, while streamlining transport and catabolic processes. Further study of the differentially expressed putative genes could provide deeper insight into the dynamic response to IB formation.

Share

COinS