TECHNOLOGIES FOR IMAGING WITH BIOLUMINESCENTLY LABELED PROBES
A DISSERTATION
SUBMITTED TO THE DEPARTMENT OF BIOENGINEERING
AND THE COMMITTEE ON GRADUATE STUDIES
OF STANFORD UNIVERSITY
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
Andreas Markus Loening
June 2006
Abstract
Luciferases, which have seen expansive employment as reporter genes in biological research, could also be used in applications where the protein itself is fused or conjugated to ligands in order to create bioluminescently-labeled imaging probes. In the context of small animal imaging, bioluminescent labeling can be expected to yield greater sensitivity than more traditional fluorescent or radioisotopic labeling approaches. This dissertation focuses on developing the underlying technologies to enable bioluminescent labeling as a routine methodology.
For a variety of practical reasons, the luciferase from Renilla reniformis is generally the most appropriate for use as a bioluminescent label. However, the native enzyme is overly labile in serum, and a consensus sequence driven mutagenesis screen was employed to improve its properties. The result of this mutagenesis was an 8 mutation variant of Renilla luciferase (RLuc8) that, compared to the parental enzyme, was 200-fold more resistant to inactivation in murine serum and exhibited a 4-fold improvement in light output. Also generated were variants optimized for use as reporter genes that showed 5-fold greater light output while exhibiting greater responsiveness to transient gene expression. An additional impediment with Renilla luciferase is that its bioluminescence emission spectrum is not ideal for in vivo imaging. Through a combination of random mutagenesis and site-directed mutations in the substrate-binding pocket, variants of Renilla luciferase with up to 60 nm red-shifts were created that are more optimal for imaging applications.
To further facilitate the analysis of Renilla luciferase, its crystallographic structure was determined to a resolution of 1.5 angstroms. This represents the first structure of a coelenterazine-dependent luciferase, and should aid future studies involving this enzyme. Additional work presented that extends beyond bioluminescent labeling includes: AMIDE, an open source software tool for displaying and analyzing multimodality volumetric image data sets; the crystallographic structure of Renilla reniformis green fluorescent protein; and bacterial expression and analysis of Gaussia princeps luciferase.