Aggregation properties of a therapeutic peptide for rheumatoid arthritis: a spectroscopic and molecular dynamics study

The biological properties of therapeutic peptides, such as their pharmacokinetics and pharmacodynamics, are correlated with their structure and aggregation properties. Herein, we studied the aggregation properties of a therapeutic peptide (CIGB-814), currently in phase 2 clinical trial, for the treatment of rheumatoid arthritis over a wide range of concentrations (μM–mM). We applied spectroscopic techniques (fluorescence, circular dichro- ism, resonance, and dynamic light scattering), atomic force microscopy, and molecular dynamics simulations to determine the aggregation mechanism of CIGB-814. We found that the hierarchical aggregation of CIGB-814 at micromolar concentrations was initiated by the formation of peptide oligomers. Subsequently, the peptide oligomers trigger the nucleation and growth of peptide nanostructures (cac = 123 μM), ultimately leading to the fibrillization of CIGB-814 (cac’ = 508 μM). These results pave the way for a deeper understanding of the CIGB-814 therapeutic activity and may give important insights on its pharmacokinetics

Scaling-up fermentation of Escherichia coli for production of recombinant P64k protein from Neisseria meningitidis

Background: P64k is a Neisseria meningitidis high molecular weight protein present in meningococcal vaccine preparations. The lpdA gene, which encodes for this protein, was cloned in Escherichia coli and the P64k recombinant protein was expressed in E. coli K12 GC366 cells under the control of a tryptophan promoter. P64k was expressed as an intracellular soluble protein about 28% of the total cellular protein. Several scale-up criteria of fermentation processes were studied to obtain the recombinant P64k protein at the pilot production scale. Results: The best operational conditions at a larger scale production of P64k recombinant protein were studied and compared using the four following criteria: Constant Reynold's number (Re constant), Constant impeller tip speed (n di constant), Constant power consumption per unit liquid volume (P/V constant) and Constant volumetric oxygen transfer coefficients (KLa/k constant). The highest production of the recombinant protein was achieved based on the constant KLa/k scale-up fermentation criterion, calculating the aeration rate (Q) and the impeller agitation speed (n) by iterative process, keeping constant the KLa/k value from bench scale. The P64k protein total production at the 50 l culture scale was 546 mg l-1 in comparison with the 284 mg l-1 obtained at 1.5 l bench scale. Conclusions: The methodology described herein, for the KLa/k scale-up fermentation criterion, allowed us to obtain the P64k protein at 50 l scale. A fermentation process for the production of P64k protein from N. meningitidis was established, a protein to be used in future vaccine formulations in humans.How to cite: Espinosa R, García J, Narciandi E, et al. Scaling-up fermentation of Escherichia coli for production of recombinant P64k protein from Neisseria meningitidis. Electron J Biotechnol 2018;33. https://doi.org/10.1016/j.ejbt.2018.03.004. Keywords: Escherichia coli, Fermentation process, Fermentation, Gram negative diplococcus, High molecular weight protein, Meningococcal vaccine, Neisseria meningitidis, Obligate human pathogen, Recombinant P64k protein, Scale-up, Tryptophan promote

Molecular cloning, expression and IgE-immunoreactivity of phospholipase A1, a major allergen from Polybia paulista (hymenoptera: vespidae) venom

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Polybia paulista (Hymenoptera: Vespidae) is a clinically relevant social wasp that frequently causes stinging accidents in southeast Brazil. To date, diagnosis and specific immunotherapy (SIT) of allergy are based on the use of crude venom extracts. Produ1244452FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIORFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)2006/54799-6; 2014/13936- 7; 2009/51539-101197/ 10-DF

Proteomic Study to Survey the CIGB-552 Antitumor Effect

CIGB-552 is a cell-penetrating peptide that exerts in vitro and in vivo antitumor effect on cancer cells. In the present work, the mechanism involved in such anticancer activity was studied using chemical proteomics and expression-based proteomics in culture cancer cell lines. CIGB-552 interacts with at least 55 proteins, as determined by chemical proteomics. A temporal differential proteomics based on iTRAQ quantification method was performed to identify CIGB-552 modulated proteins. The proteomic profile includes 72 differentially expressed proteins in response to CIGB-552 treatment. Proteins related to cell proliferation and apoptosis were identified by both approaches. In line with previous findings, proteomic data revealed that CIGB-552 triggers the inhibition of NF-κB signaling pathway. Furthermore, proteins related to cell invasion were differentially modulated by CIGB-552 treatment suggesting new potentialities of CIGB-552 as anticancer agent. Overall, the current study contributes to a better understanding of the antitumor action mechanism of CIGB-552