Current strategies for protein production and purification enabling membrane protein structural biology

Abstract

Membrane proteins are still heavily underrepresented in the protein data bank (PDB) due to multiple bottlenecks. The typical low abundance of membrane proteins in their natural hosts makes it necessary to overexpress these proteins either in heterologous systems or through in vitro translation/cell-free expression. Heterologous expression of proteins, in turn, leads to multiple obstacles due to the unpredictability of compatibility of the target protein for expression in a given host. The highly hydrophobic and/or amphipathic nature of membrane proteins also leads to challenges in producing a homogeneous, stable, and pure sample for structural studies. Circumventing these hurdles has become possible through introduction of novel protein production protocols; efficient protein isolation and sample preparation methods; and, improvement in hardware and software for structural characterization. Combined, these advances have made the past 10-15 years very exciting and eventful for the field of membrane protein structural biology, with an exponential growth in the number of solved membrane protein structures. In this review, we focus on both the advances and diversity of protein production and purification methods that have allowed this growth in structural knowledge of membrane proteins through X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryoelectron microscopy (cryo-EM).

Hydrogen-Deuterium Exchange Mass-Spectrometry of Secondary Active Transporters: From Structural Dynamics to Molecular Mechanisms

“…Indeed, while membrane proteins account for~26% of the human proteome, their structures represent only ∼2% of the structures in the Protein Data Bank (Fagerberg et al, 2010;Pandey et al, 2016). Several obstacles hamper the structural investigation of membrane proteins, requiring the development of state-of-the-art technologies for elucidating their molecular architectures (Pandey et al, 2016;Masson et al, 2017;Hagn et al, 2018;Ravula and Ramamoorthy, 2019;Redhair et al, 2019). The major obstacles are that their overexpression and purification and structural studies by most techniques (e.g., X-ray crystallography) remain limited in many cases.…”

Production and Structural Analysis of Membrane‐Anchored Proteins in Phospholipid Nanodiscs

“…

Structural studies on membrane-anchored proteins containing at ransmembrane (TM) helix have been hampered by difficulties in producing these proteins in a natively folded form. [2] This approach requires extensive screening for suitable buffers and detergents;h owever, it ofteny ields homogenous preparations of folded and functionally active membranep roteins. Thus,T Mhelicesa re removed for structural studies, which neglects the pivotal role of am embrane on protein function.

…”

Smoothing membrane protein structure determination by initial upstream stage improvements

“…The evaluation of purified protein quality is crucial in any protein production process and should be accurately performed to avoid irreproducible and misleading observations in the subsequent studies (Raynal et al 2014). After production, MP need to be efficiently solubilized (recently reviewed by Hardy et al 2018 andPopot 2018) and purified (Pandey et al 2016), from which their quality in terms of purity, homogeneity, activity, and structural conformity should be assessed (Oliveira and Domingues 2018;Raynal et al 2014). In this review, generic guidelines and host characteristics aiming an accurate choice of the host expression system that better suits particular needs will be initially overviewed in this review, and then we discuss important advances reported at the level of the upstream stage of recombinant MP production processes using E. coli, P. pastoris, and mammalian cell lines, representative of major expression systems used for protein expression.…”