Abstract
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.
Highlights
Integral membrane proteins (IMPs) are notoriously difficult to study
The splice forms synaptogyrin 1b and 1c have shortened C-termini compared to synaptogyrin 1a, synaptogyrin 2 and synaptogyrin 3, which are predicted to be in random coil conformations [40]
To gain deeper insights into structure and functional properties of human synaptogyrin we established the recombinant production of homogeneous material in E. coli
Summary
Integral membrane proteins (IMPs) are notoriously difficult to study. The lack of structural data is just one consequence of the challenges they confront us with: toxicity to their over-production host, lipid requirements for correct folding and function, detergents destabilising the IMPs and hampering the formation of well-ordered crystals [1]. TVPs have four transmembrane segments and cytoplasmically located termini, and are highly abundant components of different vesicle types [10,11,12,13] They can be grouped into three distinct families that are referred to as physins, gyrins and secretory carrier associated membrane proteins (SCAMPs). A number of mutations, insertions and deletions of the gene encoding synaptogyrin 1 were identified in schizophrenia patients suggesting that aberrant synaptogyrin 1 function may be involved in the pathogenesis of schizophrenia [31,32,33,34] Despite these findings the understanding of synaptogyrin in the synaptic vesicle cycle is far from complete. The combination of synaptogyrin construct optimisation and the usage of the recently described E. coli strain Lemo21(DE3) improved expression levels from micrograms to several milligrams per liter of E. coli culture, sufficient for structural studies. The strategy for improving eukaryotic IMP over-expression in E. coli provided here can be adapted to other IMPs
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