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Abstract: Display technologies (e.g. phage and ribosome display) are powerful tools for selecting and evolving protein binders against various target molecules. SNAP display is a DNA display technology that is conducted entirely in vitro: DNA encoding a library of variants is encapsulated in water-in-oil droplets wherein in vitro protein expression and covalent coupling to the encoding DNA occurs. Here, we explore critical factors for the successful performance of SNAP display based on a set of experiments designed to measure and quantify to what extent they affect selection efficiency. We find that, in SNAP display, the reconstituted cell free expression system PURExpress led to 1.5-fold more active protein and achieved 3.5-fold greater DNA recovery in model selections compared to the RTS 100 Escherichia coli lysate based expression system. We report on the influence parameters including droplet occupancy, valency and selection stringency have on recovery and enrichment. An improved procedure involving bivalent display and stringent selection against a model target, Her2, led to a 107-fold enrichment of a DARPin (H10-2-G3, known to bind Her2 with picomolar affinity) over a non-binding DARPin after three rounds of selection. Furthermore, when spiked into a mixture of DARPins with different affinities, DARPin H10-2-G3 outcompeted all other variants demonstrating SNAP display's ability to efficiently resolve clones with affinities in the nano- to picomolar range. These data establish SNAP display as an in vitro protein engineering tool for isolating protein binders and provide a framework for troubleshooting affinity selections. [Copyright &y& Elsevier]

Abstract: We report on the characterisation of a member of the acylaminoacyl peptidase family, the first isolated from bacteria. The enzyme was obtained from the psychrophilic bacterium Sporosarcina psychrophila and shows the typical features of cold adaptation (low Tm , optimal temperature of 40 °C, poor thermal stability). It was also tested for substrate specificity, effect of metals, temperature dependence and structure stability and revealed promiscuous catalytic activity on at least two chemically distinct substrates, with kcat /Km values for ester hydrolysis and acylamino acids cleavage of 1.7 × 104 s−1 M−1 and 6.2 × 103 s−1 M−1, respectively. Despite some properties cannot be explained with current models, results report on the relevance of structural and catalytic properties for the successful adaptation to cold temperatures. [Copyright &y& Elsevier]