24 research outputs found
In-cell NMR characterization of the secondary structure populations of a disordered conformation of Ī±-Synuclein within E. coli cells
Ī±-Synuclein is a small protein strongly implicated in the pathogenesis of Parkinsonās disease and related neurodegenerative disorders. We report here the use of in-cell NMR spectroscopy to observe directly the structure and dynamics of this protein within E. coli cells. To improve the accuracy in the measurement of backbone chemical shifts within crowded in-cell NMR spectra, we have developed a deconvolution method to reduce inhomogeneous line broadening within cellular samples. The resulting chemical shift values were then used to evaluate the distribution of secondary structure populations which, in the absence of stable tertiary contacts, are a most effective way to describe the conformational fluctuations of disordered proteins. The results indicate that, at least within the bacterial cytosol, Ī±-synuclein populates a highly dynamic state that, despite the highly crowded environment, has the same characteristics as the disordered monomeric form observed in aqueous solution
Latent analysis of unmodified biomolecules and their complexes in solution with attomole detection sensitivity
The study of biomolecular interactions is central to an understanding of function, malfunction and therapeutic modulation of biological systems, yet often involves a compromise between sensitivity and accuracy. Many conventional analytical steps and the procedures required to facilitate sensitive detection, such as the incorporation of chemical labels, are prone to perturb the complexes under observation. Here we present a 'latent' analysis approach that uses chemical and microfluidic tools to reveal, through highly sensitive detection of a labelled system, the behaviour of the physiologically relevant unlabelled system. We implement this strategy in a native microfluidic diffusional sizing platform, allowing us to achieve detection sensitivity at the attomole level, determine the hydrodynamic radii of biomolecules that vary by over three orders of magnitude in molecular weight, and study heterogeneous mixtures. We illustrate these key advantages by characterizing a complex of an antibody domain in the solution phase and under physiologically relevant conditions.We would like to thank the ERC, BBSRC, Wellcome Trust, Newman Foundation, Winston Churchill Foundation, and Elan Pharmaceuticals for financial support. E.D.G was supported by the MRC (G1002272)
Properties, production, and applications of camelid single-domain antibody fragments
Camelids produce functional antibodies devoid of light chains of which the single N-terminal domain is fully capable of antigen binding. These single-domain antibody fragments (VHHs or NanobodiesĀ®) have several advantages for biotechnological applications. They are well expressed in microorganisms and have a high stability and solubility. Furthermore, they are well suited for construction of larger molecules and selection systems such as phage, yeast, or ribosome display. This minireview offers an overview of (1) their properties as compared to conventional antibodies, (2) their production in microorganisms, with a focus on yeasts, and (3) their therapeutic applications
Production, quality control, stability, and potency of cGMP-produced Plasmodium falciparum RH5.1 protein vaccine expressed in Drosophila S2 cells
Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is a leading asexual blood-stage vaccine candidate for
malaria. In preparation for clinical trials, a full-length PfRH5 protein vaccine called āRH5.1ā was produced as a soluble product under
cGMP using the ExpreS2 platform (based on a Drosophila melanogaster S2 stable cell line system). Following development of a highproducing
monoclonal S2 cell line, a master cell bank was produced prior to the cGMP campaign. Culture supernatants were
processed using C-tag affinity chromatography followed by size exclusion chromatography and virus-reduction filtration. The
overall process yielded >400 mg highly pure RH5.1 protein. QC testing showed the MCB and the RH5.1 product met all specified
acceptance criteria including those for sterility, purity, and identity. The RH5.1 vaccine product was stored at ā80 Ā°C and is stable for
over 18 months. Characterization of the protein following formulation in the adjuvant system AS01B showed that RH5.1 is stable in
the timeframe needed for clinical vaccine administration, and that there was no discernible impact on the liposomal formulation of
AS01B following addition of RH5.1. Subsequent immunization of mice confirmed the RH5.1/AS01B vaccine was immunogenic and
could induce functional growth inhibitory antibodies against blood-stage P. falciparum in vitro. The RH5.1/AS01B was judged
suitable for use in humans and has since progressed to phase I/IIa clinical trial. Our data support the future use of the Drosophila S2
cell and C-tag platform technologies to enable cGMP-compliant biomanufacture of other novel and ādifficult-to-expressā
recombinant protein-based vaccines
Structure and properties of a complex of alpha-synuclein and a single-domain camelid antibody.
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