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Review
. 2016 Dec;94(6):507-527.
doi: 10.1139/bcb-2015-0143. Epub 2016 Jan 20.

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

Aditya Pandey  1 , Kyungsoo Shin  1 , Robin E Patterson  1 , Xiang-Qin Liu  1 , Jan K Rainey  1   2
Affiliations
Review

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

Aditya Pandey et al. Biochem Cell Biol. 2016 Dec.

Abstract

Membrane proteins are still heavily under-represented in the protein data bank (PDB), owing 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, owing 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 the 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 cryo-electron microscopy (cryo-EM).

Keywords: biologie structurale; enrichissement d’isotopes stables; expression et purification des protéines; fusion proteins; membrane protein; protein expression and purification; protéine membranaire; protéines de fusion; stable isotope enrichment; structural biology.

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Figures

Figure 1
Figure 1
Source of protein for all 2631 experimentally solved membrane protein structures currently available in the PDBTM. (Numbers based upon annotations in the PDB file headers, with “unspecified source” based only on the PDB file header.)
Figure 2
Figure 2
Expression system employed for all solved membrane protein structures produced by recombinant expression currently available in the PDBTM. (Numbers based upon annotations in the PDB file headers; hence, the total of 1719 is slightly lower than the 1855 implied by Figure 1.)
Figure 3
Figure 3
Flowchart outlining various optimization steps allowing for structural characterization of membrane proteins by XRD, NMR spectroscopy, or cryo-EM.
See this image and copyright information in PMC

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