The high-throughput production of membrane proteins

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.

Successful production of antibodies against extra cytoplasmic loops of the Mycobacterium tuberculosis ABC transporter Rv1819c

“…Similarly, efflux pumps are upregulated in bacteria during treatment with antibiotics and could become candidate tools to treat infectious diseases [26,27]. However, the use of other targets like the transmembrane permeases for these means is sparsely explored, primarily due to the complications in their production, high hydrophobicity of the TM segments necessitating the use of detergents and uncertain immunogenicity of these proteins [28,29].…”