GFP as a tool to monitor membrane protein topology and overexpression in Escherichia coli

• Main Author: Drew, David
• Format: Doctoral Thesis
• Language: English
• Published: Stockholms universitet, Institutionen för biokemi och biofysik 2005
• Subjects: GFP; membrane protein; topology; overexpression; high-throughput; Biochemistry; Biokemi
• Online Access: http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-734; http://nbn-resolving.de/urn:isbn:91-7155-160-3

Membrane proteins are essential for life, and roughly one-quarter of all open reading frames in sequenced genomes code for membrane proteins. Unfortunately, our understanding of membrane proteins lags behind that of soluble proteins, and is best reflected by the fact that only 0.5% of the structures deposited in the protein data-bank (PDB) are of membrane proteins. This discrepancy has arisen because their hydrophobicity - which enables them to exist in a lipid environment - has made them resistant to most traditional approaches used for procuring knowledge from their soluble counter-parts. As such, novel methods are required to facilitate our knowledge acquisition of membrane proteins. In this thesis a generic approach for rapidly obtaining information on membrane proteins from the classic bacterial encyclopedia Escherichia coli is described. We have developed a Green Fluorescent Protein C-terminal tagging approach, with which we can acquire information as to the topology and ‘expressibility’ of membrane proteins in a high-throughput manner. This technology has been applied to the whole E. coli inner membrane proteome, and stands as an important advance for further membrane protein research.