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Technology Topics Protein Expression

Retrospective: Producing Proteins the PSI:Biology Way

SBKB [doi:10.1038/sbkb.2015.24]
Technical Highlight - July 2015
Short description: PSI:Biology developed new expression vectors and other tools and tricks to obtain samples of its challenging protein targets.

An example of PSI-enabled cloning and expression technology: the schematic portraying globular proteins used to create GCPR chimeras necessary to crystallize the receptor. Figure from ref. 16, permission granted by Elsevier, 2015.

The Protein Structure Initiative has solved 6,900 protein structures in 15 years as a result of newly developed high-throughput (HTP) technologies. As the PSI comes to a close this month, we highlight a few of the methods and technologies developed to meet the needs of PSI:Biology targets. For a broader review of PSI results, we invite you to revisit the ∼500 research and technical reviews written by Nature Publishing Group, and the Featured Systems illustrated by David Goodsell on the SBKB's new homepage beginning in July 2015.

Research encourages us to be continually innovative. Novel HTP cloning, and expression and purification methods developed for PSI-2 were already described in detail in our 2010 PSI-2 retrospective. However, new classes of structural targets were proposed for PSI:Biology, including mammalian proteins, complexes and membrane proteins, which required brand new strategies to produce sample quantities sufficient for structure determination.

The first step, as usual, is to use an expression vector that will yield large quantities of soluble and stable proteins. This can be difficult when the target (eukaryotic) protein is from a very different organism than the host (bacterial) producer. Another complication arises when the proteins in the targeted need to be coexpressed to maintain stability and function. In response to these challenges, a new generation of bacterial expression vectors specifically for ligation-independent cloning (LIC) was designed by the PSI MCSG. Eleven of these new vectors contain additional tRNAs for rare codons of arginine and isoleucine with different purification tag options 1 . Additional vectors designed by PSI NYCOMPS improve coexpression of mammalian proteins within the preferred mammalian cell lines. 2 These new vectors, as well as all PSI expression vectors, are available from the PSI:Biology-Materials Repository at DNASU 3 .

Having the biologically appropriate construct DNA is sometimes not enough for mammalian proteins. Using experiences from their own pipeline, PSI NYSGRC researchers published a set of best-practices papers for expressing mammalian proteins 4, 5, 6 . The authors describe the appropriate cell lines to use, best vectors, transformation tips, growth conditions to test, tricks to limit (or enhance) post-translational modifications and selection methods to improve yields.

There is a different set of obstacles involved in studying membrane proteins. These proteins tend to aggregate due to their hydrophobic segments, and thus require insertion and protection in a membrane bilayer, detergents or other mimetics. These conditions often interfere with crystallization and structure determination. PSI NYCOMPS published two best-practices guides on how to optimize expression and purification of membrane proteins using pNYCOMPS LIC bacterial vectors 7, 8 . These methods, though developed in PSI-2, continued to be their main approach through PSI:Biology. A method to insert membrane protein into nanodiscs was also developed and compared to other membrane mimetics 9, 10 ; the benefits of nanodiscs include their compatibility with NMR studies 11 .

Cell-free expression systems were also shown to be a viable method for expressing different membrane proteins 12, 13, 14 . Colleagues at PSI MPSbyNMR noticed that the cell-free's simpler protein-building machinery sometimes had problems with translation initiation, and thus lower yields. To increase the probability of success, a set of “tags” was introduced during cloning that would create a variety of landing sites for the translation machinery and therefore multiple chances at achieving the right sequence 15 . After the initial screening, researchers can select the sequence that resulted in the highest yields.

Lastly, some structure determination experiments can benefit from modifications introduced during cloning and expression. For example, nearly all of the GPCR structures were actually chimeras, solved by fusing thermostable domains into their floppy but essential third loops 16 . Small globular domains, including the original lysozyme followed by T4 ligase fragment, flavodoxin, xylanase, rubredoxin and apo-Cytochrome b562RIL, not only provide the stability needed to promote crystallization but can also be used for molecular replacement to obtain phase information necessary for structure determination. PSI MCSG previously reported that reductive methylation of lysine residues can improve crystallization of unique proteins that initially failed to produce diffraction-quality crystals. More recent efforts extended those methods to include ethylation and isopropylation in the PSI MCSG protein crystallization pipeline 17 . Using the same standard procedures, crystal structures of 12 new proteins were determined, including the first ethylated and the first isopropylated protein structures.

These strategies were made possible by the “large-scale” science promoted by the PSI program. In order to explore such possibilities, countless trials and constructs needed to be tested to fine-tune these methods, an achievement that would hardly be possible within smaller research programs. The developed methodologies are now broadly available and ready to assist all scientists experiencing similar protein production problems.

Margaret Gabanyi


  1. W. H. Eschenfeldt et al. New LIC vectors for production of proteins from genes containing rare codons.
    J Struct Funct Genomics. 14, 135-144 (2013). doi:10.1007/s10969-013-9163-9

  2. Z. Assur, W. A. Hendrickson & F. Mancia Tools for coproducing multiple proteins in mammalian cells.
    Methods Mol Biol. 801, 173-187 (2012). doi:10.1007/978-1-61779-352-3_12

  3. C. Y. Seiler et al. DNASU plasmid and PSI:Biology-Materials repositories: resources to accelerate biological research.
    Nucleic Acids Res. 42, D1253-1260 (2014). doi:10.1093/nar/gkt1060

  4. S. C. Almo et al. Protein production from the structural genomics perspective: achievements and future needs.
    Curr Opin Struct Biol. 23, 335-344 (2013). doi:10.1016/

  5. S. C. Almo & J. D. Love Better and faster: improvements and optimization for mammalian recombinant protein production.
    Curr Opin Struct Biol. 26, 39-43 (2014). doi:10.1016/

  6. A. D. Bandaranayake & S. C. Almo Recent advances in mammalian protein production.
    FEBS Lett. 588, 253-260 (2014). doi:10.1016/j.febslet.2013.11.035

  7. F. Mancia & J. Love High-throughput expression and purification of membrane proteins.
    J Struct Biol. 172, 85-93 (2010). doi:10.1016/j.jsb.2010.03.021

  8. F. Mancia & J. Love High throughput platforms for structural genomics of integral membrane proteins.
    Curr Opin Struct Biol. 21, 517-522 (2011). doi:10.1016/

  9. M. Etzkorn et al. Cell-free expressed bacteriorhodopsin in different soluble membrane mimetics: biophysical properties and NMR accessibility.
    Structure. 21, 394-401 (2013). doi:10.1016/j.str.2013.01.005

  10. C. Roos et al. High-level cell-free production of membrane proteins with nanodiscs.
    Methods Mol Biol. 1118, 109-130 (2014). doi:10.1007/978-1-62703-782-2_7

  11. L. Susac, R. Horst & K. Wuthrich Solution-NMR characterization of outer-membrane protein A from E. coli in lipid bilayer nanodiscs and detergent micelles.
    Chembiochem. 15, 995-1000 (2014). doi:10.1002/cbic.201300729

  12. B. W. Jarecki, S. Makino, E. T. Beebe, B. G. Fox & B. Chanda Function of Shaker potassium channels produced by cell-free translation upon injection into Xenopus oocytes.
    SciRep. 3, 1040 (2013). doi:10.1038/srep01040

  13. C. Roos et al. Co-translational association of cell-free expressed membrane proteins with supplied lipid bilayers.
    Mol Membr Biol. 30, 75-89 (2013). doi:10.3109/09687688.2012.693212

  14. C. Boland et al. Cell-free expression and in meso crystallisation of an integral membrane kinase for structure determination.
    Cell Mol Life Sci. 71, 4895-4910 (2014). doi:10.1007/s00018-014-1655-7

  15. S. Haberstock et al. A systematic approach to increase the efficiency of membrane protein production in cell-free expression systems.
    Protein Expr Purif. 82, 308-316 (2012). doi:10.1016/j.pep.2012.01.018

  16. E. Chun et al. Fusion partner toolchest for the stabilization and crystallization of G protein-coupled receptors.
    Structure. 20, 967-976 (2012). doi:10.1016/j.str.2012.04.010

  17. K. Tan et al. Salvage of failed protein targets by reductive alkylation.
    Methods Mol Biol. 1140, 189-200 (2014). doi:10.1007/978-1-4939-0354-2_15

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