![]() Strong protein bands are observed for A2, E2 and H3 among others. Protein bands can be excised and analysed using mass spectrometry to identify the target protein or contaminants. ( Step 5) HTP characterisation using ( A) SDS–PAGE to assess protein purity, yield and susceptibility to proteases. ![]() ( Step 4) HTP purification using 96-well blocks and filter plates to purify 96 different conditions (constructs, encapsulation reagents, solvent conditions or additives) in under 12 h. ( Step 3) HTP expression enables the parallel production of all cloned constructs in a few days to 2 weeks depending on expression system. ( Step 2) HTP-cloning enables a diverse library of clones to be established in a few days. Constructs include truncations, (of mainly disordered regions and domain boundaries), functional and disease mutations, and a variety of fusion-tags. six–24 constructs are designed for each MP target. ( Step 1) Bioinformatics servers and databases such as PSIpred and Uniprot are used to aid construct design, highlighting secondary structural features, domain boundaries critical residues, PTMs and mutant and target isoforms. An illustrative overview of the HTP production of membrane proteins.Īn illustrative overview of the HTP production of membrane proteins. While HTP methods have addressed some rate-limiting production issues, the process is still largely empirical. HTP MP production has developed significantly over the last two decades and driven the deposition of over 4500 MP structures in the Protein Databank. The rapid identification of successful constructs and exclusion of those likely to fail is key to an effective approach. Many expression systems have been adapted for HTP including full automation. To place this in the context of structural biology, ∼100–250 µg of protein is required per crystallisation plate whereas 10–20 µg are required for each cryo-electron microscopy (cryo-EM) grid. ![]() Production yields vary depending on expression system, target protein and production scale but typically between 50 µg and 2 mg of purified protein can be obtained from one litre of cell culture. MPs are expressed in prokaryotic and eukaryotic hosts as well as cell-free systems. Different homologues, truncates and functional mutants should be screened to ensure success. Here we discuss different methods used for the high-throughput production of membrane proteins for structural biology.Ĭonstructing a clone that yields sufficient functional protein is the rate-limiting step affecting membrane protein (MP) production necessitating high-throughput (HTP) approaches ( Figure 1). 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. As structural techniques advance, sample requirements will change. In this way, constructs with divergent requirements can be produced for a variety of structural applications. Parameters that affect production such as expression host, membrane protein origin, expression vector, fusion-tags, encapsulation reagent and solvent composition are screened in parallel. ![]() To maximise success, high-throughput strategies were developed that rely upon simple screens to identify successful constructs and rapidly exclude those unlikely to work. However, low levels of naturally abundant protein and the hydrophobic nature of membrane proteins makes production difficult. The structural and functional characterisation of membrane proteins is therefore crucial. More than half of all therapeutics directly affect membrane protein function while nanopores enable DNA sequencing. 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.
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