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Over the decades, the bacterium Escherichia coli (E. coli) has become the cornerstone of recombinant protein production, used for heterologous synthesis of a variety of membrane proteins. Due to its rapid growth to high densities in cheap media, and its ease of manipulation and handling, E. coli is an excellent host cell for a range of membrane protein targets. Furthermore, its genetic tractability allows for a variety of gene constructs to be screened for optimal expression conditions, resulting in relatively high yields of membrane protein in a short amount of time. Here, we describe the general workflow for the production of membrane proteins in E. coli. The protocols we provide show how the gene of interest is modified, transferred to an expression vector and host, and how membrane protein yields can be optimized and analyzed. The examples we illustrate are well suited for scientists who are starting their journey into the world of membrane protein production.

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.