Insect pheromones are often derived from fatty acid metabolism. Fatty acid desaturases, enzymes introducing double bonds into fatty acids, are crucial for the biosynthesis of these chemical signals. Δ12-desaturases catalyse the biosynthesis of linoleic acid by introducing a second double bond into oleic acid, but have been identified in only a few animal species. Here, we report the functional characterisation of two Δ12-desaturases, Nvit_D12a and Nvit_D12b, from the parasitic wasp Nasonia vitripennis. We demonstrate that Nvit_D12a is expressed in the rectal vesicle of males where they produce a linoleic acid-derived sex pheromone to attract virgin females. 13 C-labelling experiments with Urolepis rufipes, a closely related species belonging to the 'Nasonia group', revealed that females, but not males, are able to synthesise linoleic acid. U. rufipes males produce an isoprenoid sex pheromone in the same gland and do not depend on linoleic acid for pheromone production. This suggests that Δ12-desaturases are common in the 'Nasonia group', but acquired a specialised function in chemical communication of those species that use linoleic acid as a pheromone precursor. Phylogenetic analysis suggests that insect Δ12-desaturases have evolved repeatedly from Δ9-desaturases in different insect taxa. Hence, insects have developed a way to produce linoleic acid independent of the omega desaturase subfamily which harbours all of the eukaryotic Δ12-desaturases known so far.

Here, we modified the multiple cloning sites from commonly used expression vectors to create a new suite of cloning plasmids that simplify and speed up cloning procedures in Escherichia coli. Each of our standardized plasmids contains two BsaI restriction sites, allowing for highly efficient cloning of genes and bringing their expression under control of either a T7 (pET21a_BsaI, pET28a_BsaI, and pMAL-c5T_BsaI) or T5 promoter (pUR22 and pUR23). Another plasmid in our suite (pTNA_BsaI) allows for generation of large gene libraries containing >108 variants, which can be constitutively expressed in selection experiments using metabolic complementation of auxotrophic E. coli strains. Coupling restriction and ligation with the BsaI restriction enzyme minimizes hands-on time, while the need for only three different primers to clone a target gene into the six different vectors keeps overall cloning costs low.

A major impediment to economical, worldwide vaccine distribution is the requirement for a “cold chain” to preserve antigenicity. We addressed this problem using a model human papillomavirus (HPV) vaccine stabilized by immobilizing HPV16 L1 capsomeres, i.e., pentameric subunits of the virus capsid, within organic glasses formed by lyophilization. Lyophilized glass and liquid vaccine formulations were incubated at 50°C for 12 weeks, and then analyzed for retention of capsomere conformational integrity and the ability to elicit neutralizing antibody responses after immunization of BALB/c mice. Capsomeres in glassy-state vaccines retained tertiary and quaternary structure, and critical conformational epitopes. Moreover, glassy formulations adjuvanted with aluminum hydroxide or aluminum hydroxide and glycopyranoside lipid A were not only as immunogenic as the commercially available HPV vaccine Cervarix®, but also retained complete neutralizing immunogenicity after high-temperature storage. The thermal stability of such adjuvanted vaccine powder preparations may thus eliminate the need for the cold chain.

Cells contain a multitude of protein complexes whose subunits interact with high specificity. However, the number of different protein folds and interface geometries found in nature is limited. This raises the question of how protein-protein interaction specificity is achieved on the structural level and how the formation of nonphysiological complexes is avoided. Here, we describe structural elements called interface add-ons that fulfill this function and elucidate their role for the diversification of protein-protein interactions during evolution. We identified interface add-ons in 10% of a representative set of bacterial, heteromeric protein complexes. The importance of interface add-ons for protein-protein interaction specificity is demonstrated by an exemplary experimental characterization of over 30 cognate and hybrid glutamine amidotransferase complexes in combination with comprehensive genetic profiling and protein design. Moreover, growth experiments showed that the lack of interface add-ons can lead to physiologically harmful cross-talk between essential biosynthetic pathways. In sum, our complementary in silico, in vitro, and in vivo analysis argues that interface add-ons are a practical and widespread evolutionary strategy to prevent the formation of nonphysiological complexes by specializing protein-protein interactions.

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