Engineering, overexpression, purification and analytical applications of recombinant bioluminescent reporters.

摘要

The initial project entailed the construction of a plasmid suitable for bacterial expression of the in vivo biotinylated aequorin photoprotein. The plasmid contained a biotin-acceptor coding sequence fused in-frame with the apoaequorin gene as well as the birA gene, encoding biotin protein ligase (BPL), responsible for post-translational biotinylation. The use of the biotin tag facilitated (a) the purification of aequorin from the crude cell extract using an avidin resin and (b) the direct complexation of aequorin with streptavidin for utilization as a reporter molecule. Streptavidin-biotinylated aequorin complexes were used for the quantification of biotinylated hybrids in a hybridization assay, with high detectability and reproducibility. Yields of in vivo biotinylated aequorin from one liter of culture were sufficient for 300 000 hybridization assays. The goal of the second project was to create novel aequorin photoproteins with improved luminescent activity over the native protein, through directed molecular evolution of the apoaequorin gene. Random mutagenesis was accomplished through repeated rounds of DNA shuffling, cloning and expression of mutant proteins, followed by screening and selection of regenerated mutants. Increased ratios of luminescent signals to the mass of total soluble protein were compared as criteria for mutant selection after five rounds of shuffling. Initial data indicated three mutants exhibited 20--60 fold higher luminescence in crude samples. Comparisons utilizing purified proteins indicated the mutant aequorins had identical luminescent activity to the native protein, and selection possibly favoured mutations allowing improved solubility of the overexpressed protein during extraction rather than higher luminescence quantum yield. The final project involved the Gaussia princeps luciferase (GLuc), which has recently been cloned as the smallest known coelenterazine-using luciferase. Presently, there are no analytical studies using GLuc as a bioluminescent reporter. Beginning with GLuc cDNA, a plasmid encoding a biotin acceptor peptide-GLuc fusion protein was constructed. This allowed overexpression in E. coli and purification by avidin affinity chromatography. The light emission of purified Gluc was studied at various coelenterazine concentrations to establish the detectability of the new reporter, which was found to reach attomole levels. Furthermore, the complexation of biotinylated GLuc with streptavidin was used as a detection reagent in a microtiter well-based, bioluminometric DNA hybridization assay. Source: Dissertation Abstracts International, Volume: 64-01, Section: B, page: 0196. Adviser: K. Taylor. Thesis (Ph.D.)--University of Windsor (Canada), 2002.