PepT1 and PepT2 are major facilitator superfamily (MFS) transporters that utilize a proton gradient to drive the uptake of di‐ and tri‐peptides in the small intestine and kidney, respectively. They are the major routes by which we absorb dietary nitrogen and many orally administered drugs. Here, we present the crystal structure of PepTSo, a functionally similar prokaryotic homologue of the mammalian peptide transporters from Shewanella oneidensis . This structure, refined using data up to 3.6 Å resolution, reveals a ligand‐bound occluded state for the MFS and provides new insights into a general transport mechanism. We have located the peptide‐binding site in a central hydrophilic cavity, which occludes a bound ligand from both sides of the membrane. Residues thought to be involved in proton coupling have also been identified near the extracellular gate of the cavity. Based on these findings and associated kinetic data, we propose that PepTSo represents a sound model system for understanding mammalian peptide transport as catalysed by PepT1 and PepT2. There is a [Have You Seen?][1] (July 2012) linked with this article. [1]: http://dx.doi.org/10.1038/emboj.2012.206

Self-assembling peptide-based biomaterials are being developed for use as 3D tissue engineering scaffolds and for therapeutic drug-release applications. Chemical synthesis provides custom-made peptides in small quantities, but production approaches based upon transgenic organisms might be more cost-effective for large-scale peptide production. Long lead times for developing appropriate animal clones or plant lines and potential negative public opinion are obstacles to these routes. Microbes, particularly safe organisms used in the food industry, offer a more rapid route to the large-scale production of recombinant self-assembling biomaterials. In this review, recent advances and challenges in the recombinant production of collagen, elastin and de novo designed self-assembling peptides are discussed.

The cysteine proteinase inhibitor, oryzacystatin‐I (Oc‐I), and several engineered Oc‐I variants have been tested for efficacy in inhibiting growth and development of both the free‐living nematode, Caenorhabditis elegans, and the plant parasitic nematode Globodera pallida. To assist in the design of protein engineering experiments to improve the efficacy of Oc‐I, an alignment of 28 cystatins and a molecular model of Oc‐I were generated. Inhibitory activities (K_i) of wild‐type and variant forms of Oc‐I against both papain and the C. elegans cysteine proteinase, gcp‐1, were measured. For one variant, in which residue Asp86 was deleted (Oc‐IΔD86), the K,i was reduced by 13‐ to 14‐fold. LD₅₀ studies to test the effect of Oc‐I and Oc‐IΔD86 against C. elegans showed the relative median potency of Oc‐IΔD86 to be 0.76 that of wild‐type Oc‐I. When expressed in tomato hairy roots both Oc‐I and Oc‐IΔD86 had a detrimental effect on growth and development of G. pallida. This effect was significantly greater on Oc‐IΔD86‐expressing roots leading to a reduction in size of G. pallida females to a level at which fecundity is profoundly affected.

Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications.