Site-specific protein modification using immobilized sortase in batch and continuous-flow systems

Transpeptidation catalyzed by ​sortase A allows the preparation of proteins that are site-specifically and homogeneously modified with a wide variety of functional groups, such as fluorophores, PEG moieties, lipids, glycans, bio-orthogonal reactive groups and affinity handles. This protocol describes immobilization of ​sortase A on a solid support (Sepharose beads). Immobilization of ​sortase A simplifies downstream purification of a protein of interest after labeling of its N or C terminus. Smaller batch and larger-scale continuous-flow reactions require only a limited amount of enzyme. The immobilized enzyme can be reused for multiple cycles of protein modification reactions. The described protocol also works with a Ca²⁺-independent variant of ​sortase A with increased catalytic activity. This heptamutant variant of ​sortase A (7M) was generated by combining previously published mutations, and this immobilized enzyme can be used for the modification of calcium-senstive substrates or in instances in which low temperatures are needed. Preparation of immobilized ​sortase A takes 1–2 d. Batch reactions take 3–12 h and flow reactions proceed at 0.5 ml h⁻¹, depending on the geometry of the reactor used.United States. National Institutes of Health (RO1 AI087879

25th Annual Computational Neuroscience Meeting: CNS-2016

Abstracts of the 25th Annual Computational Neuroscience Meeting: CNS-2016 Seogwipo City, Jeju-do, South Korea. 2–7 July 201

Effect of frequency on pressure cost of ventilation and gas exchange in newborns receiving high-frequency oscillatory ventilation

BackgroundWe hypothesized that ventilating at the resonant frequency of the respiratory system optimizes gas exchange while limiting the mechanical stress to the lung in newborns receiving high-frequency oscillatory ventilation (HFOV). We characterized the frequency dependence of oscillatory mechanics, gas exchange, and pressure transmission during HFOV.MethodsWe studied 13 newborn infants with a median (interquartile range) gestational age of 29.3 (26.4-30.4) weeks and body weight of 1.00 (0.84-1.43) kg. Different frequencies (5, 8, 10, 12, and 15 Hz) were tested, keeping carbon dioxide diffusion coefficient (DCO2) constant. Oscillatory mechanics and transcutaneous blood gas were measured at each frequency. The attenuation of pressure swings (ΔP) from the airways opening to the distal end of the tracheal tube (TT) and to the alveolar compartment was mathematically estimated.ResultsBlood gases were unaffected by frequency. The mean (SD) resonant frequency was 16.6 (3.5) Hz. Damping of ΔP increased with frequency and with lung compliance. ΔP at the distal end of the TT was insensitive to frequency, whereas ΔP at the peripheral level decreased with frequency.ConclusionThere is no optimal frequency for gas exchange when DCO2 is held constant. Greater attenuation of oscillatory pressure at higher frequencies offers more protection from barotrauma, especially in patients with poor compliance.Pediatric Research advance online publication, 26 July 2017; doi:10.1038/pr.2017.151

Going public: reflections on predicaments and possibilities in public research and scholarship

On the afternoon of 14 May 2011, only minutes before a Paris-bound flight departed from Kennedy Airport, officers from the Port Authority of New York and New Jersey boarded the airplane and arrested Dominique Strauss-Kahn, former head of the International Monetary Fund (IMF). Several hours earlier, Naffisatou Diallo — a hotel housekeeper and asylee1 from Guinea — had reported that Strauss-Kahn had sexually assaulted and attempted to rape her when she went to clean the Manhattan hotel room where he had been staying until earlier that day. Almost instantaneously, news media outlets began salivating over the sordid details of the alleged scandal involving such a high-profile figure as ‘DSK’, as he is known (Goldfarb, 2011), the media initially, sympathising with Diallo (Dickey and Solomon, 2011). Before long, however, attention turned to scepticism as private details and uncertainties about Diallo’s character and her past were catapulted into full view as fodder for public consumption. Headlines splashed accounts of seemingly shady connections, questionable financial transactions, and alleged mistruths and misdeeds by the woman who brought charges against DSK (Italiano, 2011). Many of the news stories and exposés replayed what Welch and Schuster (2005) have described as the ‘noisy’ discursive construction of asylum seekers. This account draws on Cohen’s (2002) discussion of moral panic theory

Facile Site-Specific Multiconjugation Strategies in Recombinant Proteins Produced in Bacteria.

For biomedical applications, proteins may require conjugation to small and large molecules. Typical examples are dyes for imaging, cytotoxic effector molecules for cell killing, or half-life extension modules for optimized pharmacokinetics. Although many conjugation strategies are straightforward to apply, most of them do not enable site-specific and orthogonal conjugation, and do not yield a defined stoichiometry. Moreover, techniques offering these desirable features often rely on complex expression procedures and suffer from low production yields. A more promising manufacturing strategy for flexible, site-specific and stoichiometrically defined payloading of proteins is the combination of click chemistry and thiol-maleimide conjugation, which even enables dual labeling when used consecutively. Here, we describe as an example the production of Designed Ankyrin Repeat Proteins (DARPins), a non-IgG binding scaffold, in a specific E. coli strain to obtain high yields of protein carrying both a thiol and an azide group. We provide straightforward protocols for strain-promoted azide-alkyne cycloaddition (SPAAC) and thiol-maleimide conjugation, and furthermore compare these conjugation chemistries with existing alternatives like copper-catalyzed azide-alkyne cycloaddition (CuAAC). Finally, detailed instructions for reactivity analysis and yield estimations of the reactions are provided