Development of High Throughput Screening Methods to Identify Potential Therapeutic Oxygen Carriers Open Access

Ge, Lan (2013)

Permanent URL: https://etd.library.emory.edu/concern/etds/736664916?locale=en
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Abstract

Blood shortage has become a worldwide problem in recent years, caused by the increased demand for blood transfusions and reduced supply of healthy blood. To address this problem, hemoglobin-based blood substitutes have been previously designed, but challenges still remain due to their instability under physiological conditions. Recently, a class of heme proteins, Heme-Nitric oxide/OXygen binding domains from Thermoanaerobacter tengcongensis (Tt H-NOX), with tunable oxygen affinity and extraordinary stability has been considered as a promising candidate for therapeutic oxygen delivery. Previous studies have focused on engineering the oxygen binding affinity of Tt H-NOX by performing site directed mutagenesis on its active site. However, given limited knowledge beyond the active site and the time required for characterization, more effective methods need to be developed for blood substitute development. Therefore, directed evolution and high throughput screening approach was developed in this study. To remove non-heme bound proteins potentially evolved from directed evolution, a high throughput heme incorporation assay was developed, which select for heme-bound proteins in whole cells. In addition, multiple optical based medium throughput oxygen affinity assays were developed. By correlating the oxygen dissociation constant of the protein with the luminescence or fluorescence output, the protein's oxygen affinity were optically measured in 96-well plates. To validate these developed assays, a site saturation library of Tt H-NOX was generated at Y140, L144, G71 and G143 sites, which can be screened for useful properties for therapeutic oxygen delivery. These studies will also highlight residues outside the heme active site that are important for modulating oxygen affinity.

Table of Contents

Table of Contents

Introduction..................1

Blood Shortage............1

Heme Proteins............1

The H-NOX Family of Heme Proteins............3

Thermoanaerobacter tengcongensis (Tt) H-NOX............4

Results and Disccusion..................10

Tt H-NOX Protein Characterization............10

Site Saturation Library Generation............11

Development of High Throughput Heme Incorporation Assay............12

Development of Oxygen Affinity Screening Assay............19

Luciferase Assay............20

HRP Assay............28

Conclusion and Future Directions..................36

Experimental Methods..................38

References..................46

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