Membrane-based inverse-transition purification facilitates a rapid isolation of various spider-silk elastin-like polypeptide fusion proteins from extracts of transgenic tobacco.

Plants can produce complex pharmaceutical and technical proteins. Spider silk proteins are one example of the latter and can be used, for example, as compounds for high-performance textiles or wound dressings. If genetically fused to elastin-like polypeptides (ELPs), the silk proteins can be reversibly precipitated from clarified plant extracts at moderate temperatures of ~ 30 °C together with salt concentrations > 1.5 M, which simplifies purification and thus reduces costs. However, the technologies developed around this mechanism rely on a repeated cycling between soluble and aggregated state to remove plant host cell impurities, which increase process time and buffer consumption. Additionally, ELPs are difficult to detect using conventional staining methods, which hinders the analysis of unit operation performance and process development. Here, we have first developed a surface plasmon resonance (SPR) spectroscopy-based assay to quantity ELP fusion proteins. Then we tested different filters to prepare clarified plant extract with > 50% recovery of spider silk ELP fusion proteins. Finally, we established a membrane-based purification method that does not require cycling between soluble and aggregated ELP state but operates similar to an ultrafiltration/diafiltration device. Using a data-driven design of experiments (DoE) approach to characterize the system of reversible ELP precipitation we found that membranes with pore sizes up to 1.2 µm and concentrations of 2-3 M sodium chloride facilitate step a recovery close to 100% and purities of > 90%. The system can thus be useful for the purification of ELP-tagged proteins produced in plants and other hosts.

Optimization for synthesis of silver nanoparticles through response surface methodology using leaf extract of Boswellia sacra and its application in antimicrobial activity.

In the present work, leaf extract of Boswellia sacra was used as reductant for synthesis of silver nanoparticles (AgNPs). The variables such as volume of Boswellia sacra leaf extract (1%), volume of silver nitrate (1 mM), and temperature were optimized by response surface methodology via Box-Behnken design for the synthesis of AgNPs. Design-Expert software generated the optimum conditions for the highest yield of silver nanoparticles as 8 mL of 1 mM AgNO3, 8 mL of 1% Boswellia sacra leaf extract, and temperature = 55 °C. The formed AgNPs were isolated and purified by centrifugation process using ethanol/ distilled water. AgNPs were characterized using FTIR, SEM, TEM, EDX, and XRD. AgNPs showed surface plasmon resonance absorption band at 422 nm. XRD pattern indicated the crystalline nature of the particles (diameter 11.17 to 37.50 nm) with face-centered cubic structure. SEM and TEM images highlighted the formation of spherical AgNPs. The energy dispersive spectroscopic spectrum confirmed the presence of elemental silver. The microbial activity of AgNPs was evaluated against bacteria and fungi. Synthesized AgNPs were very effective against Gram-positive E. coli bacterial strains and fungal strains (Penicillium chrysogenum).

Discovery of the first small-molecule CsrA-RNA interaction inhibitors using biophysical screening technologies.

AIM: CsrA is a global post-transcriptional regulator protein affecting mRNA translation and/or stability. Widespread among bacteria, it is essential for their full virulence and thus represents a promising anti-infective drug target. Therefore, we aimed at the discovery of CsrA-RNA interaction inhibitors. Results & methodology: We followed two strategies: a screening of small molecules (A) and an RNA ligand-based approach (B). Using surface plasmon resonance-based binding and fluorescence polarization-based competition assays, (A) yielded seven small-molecule inhibitors, among them MM14 (IC50 of 4 µM). (B) resulted in RNA-based inhibitor GGARNA (IC50 of 113 µM). CONCLUSION: The first small-molecule inhibitors of the CsrA-RNA interaction were discovered exhibiting micromolar affinities. These hits represent tools to investigate the effects of CsrA-RNA interaction inhibition on bacterial virulence.