Expression of eukaryotic membrane proteins in eukaryotic and prokaryotic hosts

The production of membrane proteins of high purity and in satisfactory yields is crucial for biomedical research. Due to their involvement in various cellular processes, membrane proteins have increasingly become some of the most important drug targets in modern times. Therefore, their structural and functional characterization is a high priority. However, protein expression has always been more challenging for membrane proteins than for soluble proteins. In this review, we present four of the most commonly-used expression systems for eukaryotic membrane proteins. We describe the benefits and drawbacks of bacterial, yeast, insect and mammalian cells. In addition, we describe the different features (growth rate, yield, post-translational modifications) of each expression system, and how they are influenced by the construct design and modifications of the target gene. Cost-effective and fast-growing E. coli is mostly selected for the production of small, simple membrane proteins that, if possible, do not require post-translational modifications but has the potential for the production of bigger proteins as well. Yeast hosts are advantageous for larger and more complex proteins but for the most complex ones, insect or mammalian cells are used as they are the only hosts able to perform all the post-translational modifications found in human cells. A combination of rational construct design and host cell choice can dramatically improve membrane protein production processes

Detergent-Free Membrane Protein Purification Using SMA Polymer

One of the big challenges for the study of structure and function of membrane proteins is the need to extract them from the membrane. Traditionally this was achieved using detergents which disrupt the membrane and form a micelle around the protein, but this can cause issues with protein function and/or stability. In 2009 an alternative approach was reported, using styrene maleic acid (SMA) copolymer to extract small discs of lipid bilayer encapsulated by the polymer and termed SMALPs (SMA lipid particles). Since then this approach has been shown to work for a range of different proteins from many different expression systems. It allows the extraction and purification of a target protein while maintaining a lipid bilayer environment. Recently this has led to several new high-resolution structures and novel insights to function. As with any method there are some limitations and issues to be aware of. Here we describe a standard protocol for preparation of the polymer and its use for membrane protein purification, and also include details of typical challenges that may be encountered and possible ways to address those

Use of Membrane Proteins as Antifungal Drug Targets

Fungal infections represent a much-overlooked threat that has yet to receive its due consideration. Their invasive branch has an underestimated impact on human morbidity and mortality. Despite this, research on antifungal therapies has been stalling for almost two decades now, while resistant strains have emerged on essentially every class of drugs that has been commercially available. Therefore, developing ways to counteract this emerging resistance is of paramount importance if we wish to remain capable of treating invasive fungal infections. New classes of drugs using new mechanisms of action would be highly desirable, especially if they are targeting fungal markers that have not been identified as potential targets before. This thesis project has been carried out in partnership with F2G Ltd, Manchester, UK, regarding the expression of new potential drug targets for antifungal treatments. It focuses on membrane proteins, which are a crucial gateway to the cell and an important source of untargeted markers that could represent very promising alternatives. Two main targets, both enzymatic membrane proteins, have been expressed in Pichia Pastoris yeast cells and solubilised using poly (styrene-co-maleic acid) lipid particles or SMALPs, which enables to retain the membrane protein with its surrounding lipids so that the protein stays in its native conformation. This allowed the protein to remain functional, so that a functional assay could be developed later on in order to test its activity. The final step was to test potential antifungal compounds developed by F2G to inhibit the enzymatic reaction, which was the key for the antifungal activity detected in earlier studies by F2G. Because membrane proteins are much harder to work with than soluble ones, they can sometimes be more difficult to obtain in sufficient amount and purity. Therefore, an attempt at engineering a contaminant-free P. pastoris cell line was carried out, with the goal of removing the main contaminant found during membrane protein production and purification

Detergent-Free Membrane Protein Purification Using SMA Polymer

One of the big challenges for the study of structure and function of membrane proteins is the need to extract them from the membrane. Traditionally this was achieved using detergents which disrupt the membrane and form a micelle around the protein, but this can cause issues with protein function and/or stability. In 2009 an alternative approach was reported, using styrene maleic acid (SMA) copolymer to extract small discs of lipid bilayer encapsulated by the polymer and termed SMALPs (SMA lipid particles). Since then this approach has been shown to work for a range of different proteins from many different expression systems. It allows the extraction and purification of a target protein while maintaining a lipid bilayer environment. Recently this has led to several new high-resolution structures and novel insights to function. As with any method there are some limitations and issues to be aware of. Here we describe a standard protocol for preparation of the polymer and its use for membrane protein purification, and also include details of typical challenges that may be encountered and possible ways to address those