Efficient Production of Recombinant Membrane Proteins (459-501)

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In this section, we will develop methods to enhance membrane protein and membrane protein complex production in both prokaryotic and eukaryotic expression systems. For each target, we will use techniques to mutagenize the target sequence itself, or we will mutagenize the host cells in which each target is produced. Initially, we will use chemical mutagenesis for host cells, and error-prone amplification to mutagenize targets. Selection for improved expression will be based on two different screens, one visual using green fluorescent protein (GFP), and one based on selection through antibiotic resistance. For protein complexes, we will make use of vectors with internal ribosome entry sites (IRES) for the coexpression of each target associated with a spectrally distinct marker (such as GFP or YFP), which will be used for selection. The use of spectrally distinct markers allows for the selection of cells highly expressing all components of a complex. Finally, we will use targets directly attached to different fluorophores for tracking and selection of stable complexes using chromatographic techniques.