Editorial overview: New constructs and expression of proteins Rob Meijers and Anastassis Perrakis Current Opinion in Structural Biology 2016, 38:v–vi For a complete overview see the Issue Available online 9th August 2016 http://dx.doi.org/10.1016/j.sbi.2016.07.013 0959-440X/# 2016 Elsevier Ltd. All rights reserved. Recent advances in structural biology, spearheaded by developments in electron microscopy, have led to the structural characterization of proteins and complexes that were inaccessible until recently. This puts new chal- lenges towards protein sample preparation, because the old adage still holds that a structure is no better than the sample it was prepared from. Peering through the Looking Glass, you can say that Alice the grad student has to run a little harder to catch up with the Red Queen of technical innovation. Fortunately, there are exciting technical advances to report in the field of protein sample production. In this issue we highlight a number of approaches that open up new opportunities to obtain structural information on macromolecules and assemblies that were inaccessible until recently. A development that is gaining considerable popularity in protein expression experiments is the use of gene synthesis. Reduced costs and literally single- click ordering have a major impact on throughput, quality and quantity of heterologous expression of recombinant genes. Parret et al. review how gene synthesis can boost expression of specific proteins, arguing that codon optimization is a powerful tool that needs to be exploited to its full potential. Exciting new insights in protein translation regulation should be incorpo- rated in gene synthesis to further increase expression, while avoiding the production of misfolded or insoluble proteins. Surprisingly, it appears that the most important advance may come from a redesign of expression vectors, to incorporate new insights into protein translation initiation. Introducing the gene (or genes) of interest to the appropriate DNA vehicle has been largely catalysed by the advent of Structural Genomics. The turn of the millennium has seen the evolution of a variety of cloning methods allowing the expression of proteins in various organisms. Celie et al. review the most popular methods available for that task: classic restriction and ligation dependent technologies, ligation-independent, and recombina- tion-dependent technologies. They also touch upon techniques for DNA mutagenesis and for the co-expression of more than one gene in the same host-cell. Albeit prokaryotic expression hosts remain popular, cells from higher organisms are often essential for success in expressing specific proteins or complexes. Baculovirus infected insect cells have long been established, and recent work that has been reviewed in previous editions of this series, has strengthened both the potential and the ease of use of this system, making it especially suitable for multi-protein complexes. The use of mammalian cells for the high level production of recombinant proteins is especially powerful Rob Meijers Structural Biology, EMBL Hamburg, Hamburg, Germany e-mail: r.meijers@embl-hamburg.de Rob Meijers studied chemistry at the University of Amsterdam, where he also obtained his PhD in protein crystallography. He did postdoctoral work at the Dana Farber Cancer Institute in the group of Jia-Huai Wang. After a sojourn as a staff scientist at the Soleil Synchrotron, he became a group leader at EMBL Hamburg in 2009. There he established a sample preparation and characterization (SPC) facility for synchrotron users and developed screens and protocols for protein optimization such as the RUBIC screens. His research is focused on receptors involved in wiring the brain, which made him realize we still need better technologies in sample production to capture the molecules small and large that contribute to this process. Anastassis Perrakis Department of Biochemistry, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands e-mail: a.perrakis@nki.nl Anastassis (Tassos) Perrakis studied Biology in Athens, and received a PhD in chemistry from the University of York, for research performed at EMBL-Hamburg. Following an EMBO fellowship and an appointment at the EMBL-Grenoble, he started a research group at the Netherlands Cancer Institute in 2001. His contributions in methods for X-ray crystallography (ARP/wARP and PDB_REDO) are somewhat ironically more visible than these in protein expression methods (NKI-LIC vectors, PACT screen, ProteinCCD). To complicate things further, his passion remains to understand how enzymes manage to be at the right place at the right time to do their job inside or outside the cell. Available online at www.sciencedirect.com ScienceDirect www.sciencedirect.com Current Opinion in Structural Biology 2016, 38:v–vi