Abstract
Membrane proteins, found at the junctions between the outside world and the inner workings of the cell, play important roles in human disease and are used as biosensors. More than half of all therapeutics directly affect membrane protein function while nanopores enable DNA sequencing. The structural and functional characterisation of membrane proteins is therefore crucial. However, low levels of naturally abundant protein and the hydrophobic nature of membrane proteins makes production difficult. To maximise success, high-throughput strategies were developed that rely upon simple screens to identify successful constructs and rapidly exclude those unlikely to work. Parameters that affect production such as expression host, membrane protein origin, expression vector, fusion-tags, encapsulation reagent and solvent composition are screened in parallel. In this way, constructs with divergent requirements can be produced for a variety of structural applications. As structural techniques advance, sample requirements will change. Single-particle cryo-electron microscopy requires less protein than crystallography and as cryo-electron tomography and time-resolved serial crystallography are developed new sample production requirements will evolve. Here we discuss different methods used for the high-throughput production of membrane proteins for structural biology.
Highlights
Constructing a clone that yields sufficient functional protein is the rate-limiting step affecting membrane protein (MP) production necessitating high-throughput (HTP) approaches (Figure 1)
Production yields vary depending on expression system, target protein and production scale but typically between 50 mg and 2 mg of purified protein can be obtained from one litre of cell culture
To place this in the context of structural biology, ∼100–250 mg of protein is required per crystallisation plate whereas 10–20 mg are required for each cryo-electron microscopy grid
Summary
Constructing a clone that yields sufficient functional protein is the rate-limiting step affecting membrane protein (MP) production necessitating high-throughput (HTP) approaches (Figure 1). MPs are expressed in prokaryotic and eukaryotic hosts as well as cell-free systems [1,2,3]. (Step 3) HTP expression enables the parallel production of all cloned constructs in a few days to 2 weeks depending on expression system.
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