Optimisation of Over-Expression in E. coli and Biophysical Characterisation of Human Membrane Protein Synaptogyrin 1

Progress in functional and structural studies of integral membrane proteins (IMPs) is lacking behind their soluble counterparts due to the great challenge in producing stable and homogeneous IMPs. Low natural abundance, toxicity when over-expressed and potential lipid requirements of IMPs are only a few reasons for the limited progress. Here, we describe an optimised workflow for the recombinant over-expression of the human tetraspan vesicle protein (TVP) synaptogyrin in Escherichia coli and its biophysical characterisation. TVPs are ubiquitous and abundant components of vesicles. They are believed to be involved in various aspects of the synaptic vesicle cycle, including vesicle biogenesis, exocytosis and endocytotic recycling. Even though TVPs are found in most cell types, high-resolution structural information for this class of membrane proteins is still missing. The optimisation of the N-terminal sequence of the gene together with the usage of the recently developed Lemo21(DE3) strain which allows the balancing of the translation with the membrane insertion rate led to a 50-fold increased expression rate compared to the classical BL21(DE3) strain. The protein was soluble and stable in a variety of mild detergents and multiple biophysical methods confirmed the folded state of the protein. Crosslinking experiments suggest an oligomeric architecture of at least four subunits. The protein stability is significantly improved in the presence of cholesteryl hemisuccinate as judged by differential light scattering. The approach described here can easily be adapted to other eukaryotic IMPs

Strategies for protein coexpression in Escherichia coli

E. coli is a convenient host for heterologous protein expression. Its advantages include high levels of heterologous gene expression and scalability of experiments, low cost, fast growth, a lack of posttranslational modification and an ability to express labeled (isotope or seleno-methionine) proteins. However, heterologous gene expression in E. coli can lead to the production of insoluble and/or nonfunctional target proteins. This is often due to the absence of cofactors or post-translational modifications required for function, stability or folding. Coexpression of multiple genes in E. coli, such as the members of a stable multiprotein complex1 or a protein with a chaperone2,3, can in many cases alleviate these problems. Coexpression involves the transformation of E. coli with several plasmids that have compatible origins of replication and independent antibiotic selection for maintenance. The Duet (Novagen) vectors have two multiple cloning sites per vector, five compatible origins of replication and four antibiotic selection markers, allowing the simultaneous expression of up to eight proteins. The combination of Duet vectors with other commercial plasmids allows the use of affinity tags, such as glutathione S-transferase (GST) or maltose binding protein (MBP), which can ease the recovery and improve the solubility of the desired target. Coexpression in E. coli therefore provides a useful alternative to the complicated and expensive expression systems, such as yeast, baculovirus or mammalian cell culture, which are commonly used to overcome problems of heterologous protein expression. A summary of the method is presented in Figure 1