6,053 research outputs found
Optimized rapid production of recombinant secreted proteins in CHO cells grown in suspension: The case of RBD
No full textChinese Hamster Ovary cells (CHO) have become the most common workhorse for the commercial production of therapeutic proteins, as well as for the production of recombinant proteins for biomedical research. The ability to grow at high density in suspension, the adaptability to serum free media, and the ease transfection and scale up, made CHO cell line highly productive and robust for large-scale production. Here, we present an optimized workflow used to successfully express and purify a number of human proteins with a yield up to 5 mg/L of culture. The entire protocol, from the synthetic gene design to the assessment of purified protein quality, can be completed in 2 weeks. The established cell culture platform has been efficiently adapted to rapidly produce the receptor-binding domain (RBD) in SARS-CoV-2 S protein, a protein required by many laboratories in 2020 to better understand the initial step of infection related to COVID-19 pandemic. An overall yield of 2 mg of high quality soluble RBD per liter of culture was obtained, a production 10-times cheaper than commercial preparations, this representing an intriguing strategy for future challenges
Protein engineering of d-amino acid oxidase: design of new activities for industrial and medical applications.
No full textmissin
A novel biosensor for detection of all D-amino acids based on optimized D-amino acid oxidase
No full textCharacterization and Investigation of Redox-Sensitive Liposomes for Gene Delivery
No full textA number of smart nonviral gene delivery vectors relying on bioresponsiveness have been introduced in the past few years to overcome the limits of the first generation of gene carriers. Among them, redox-sensitive lipidic and polymeric vectors exploit the presence of disulfide bonds in their structure to take advantage of the highly reductive intracellular milieu and to promote complex unpacking and nucleic acids release after cellular uptake (disulfide linker strategy). Glutathione (GSH) has been often identified as the leading actor in the intracellular reduction of bioreducible vectors but their actual mechanisms of action have been rarely investigated in depth and doubts about the real effectiveness of the disulfide linker strategy have been raised. Herein, we outline a simple protocol for the preparation and investigation of nano-sized reducible cationic liposomes, focusing on their thorough characterization and optimization as gene delivery vectors. In addition, we carefully describe the techniques and procedures necessary for the assessment of the bioreducibility of the vectors and to demonstrate that the GSH-mediated intracellular cleavage of disulfide bonds is a pivotal step in their transfection process. Liposomes composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), and of the reducible cationic lipid SS14 are reported as a practical example but the proposed protocol can be easily shifted to other formulations of reducible lipids/liposomes and to reducible polymers
Encuentros con Elena Poniatowska
No full textThe author analyzes testimonial literature from the perspective of female literature through his meeting with Elena Poniatowska. An analysis of reality vs. Fiction in Elena, Jesusa and Tinisima.El autor analiza, desde su encuentro con Elena Poniatowska, la vertiente de la literatura testimonial como literatura de mujeres. Un análisis interior de la relación entre realidad y ficción, entre Elena, Jesusa o Tinísima
Autosomal recessive cerebellar ataxias: a diagnostic classification approach according to ocular features
No full textAutosomal recessive cerebellar ataxias (ARCAs) are a heterogeneous group of neurodegenerative disorders affecting primarily the cerebellum and/or its afferent tracts, often accompanied by damage of other neurological or extra-neurological systems. Due to the overlap of clinical presentation among ARCAs and the variety of hereditary, acquired, and reversible etiologies that can determine cerebellar dysfunction, the differential diagnosis is challenging, but also urgent considering the ongoing development of promising target therapies. The examination of afferent and efferent visual system may provide neurophysiological and structural information related to cerebellar dysfunction and neurodegeneration thus allowing a possible diagnostic classification approach according to ocular features. While optic coherence tomography (OCT) is applied for the parametrization of the optic nerve and macular area, the eye movements analysis relies on a wide range of eye-tracker devices and the application of machine-learning techniques. We discuss the results of clinical and eye-tracking oculomotor examination, the OCT findings and some advancing of computer science in ARCAs thus providing evidence sustaining the identification of robust eye parameters as possible markers of ARCAs. Copyright © 2024 Lopergolo, Rosini, Pretegiani, Bargagli, Serchi and Rufa
Assays of D-amino acid oxidase activity
No full textD-amino acid oxidase (DAAO) is a well-known flavoenzyme that catalyzes the oxidative FAD-dependent deamination of D-amino acids. As a result of the absolute stereoselectivity and broad substrate specificity, microbial DAAOs have been employed as industrial biocatalysts in the production of semi-synthetic cephalosporins and enantiomerically pure amino acids. Moreover, in mammals, DAAO is present in specific brain areas and degrades D-serine, an endogenous coagonist of the N-methyl-D-aspartate receptors (NMDARs). Dysregulation of D-serine metabolism due to an altered DAAO functionality is related to pathological NMDARs dysfunctions such as in amyotrophic lateral sclerosis and schizophrenia. In this protocol paper, we describe a variety of direct assays based on the determination of molecular oxygen consumption, reduction of alternative electron acceptors, or α-keto acid production, of coupled assays to detect the hydrogen peroxide or the ammonium production, and an indirect assay of the α-keto acid production based on a chemical derivatization. These analytical assays allow the determination of DAAO activity both on recombinant enzyme preparations, in cells, and in tissue samples
The Symmetric Active Site of Enantiospecific Enzymes
No full textBiomolecules are frequently chiral compounds, existing in enantiomeric forms. Amino acids represent a meaningful example of chiral biological molecules. Both L- and D-amino acids play key roles in the biochemical structure and metabolic processes of living organisms, from bacteria to mammals. In this review, we explore the enantiospecific interaction between proteins and chiral amino acids, introducing theoretical models and describing the molecular basis of the ability of some of the most important enzymes involved in the metabolism of amino acids (i.e., amino acid oxidases, dehydrogenases, and aminotransferases) to discriminate the opposite enantiomers. Our analysis showcases the power of natural evolution in shaping biological processes. Accordingly, the importance of amino acids spurred nature to evolve strictly enantioselective enzymes both through divergent evolution, starting from a common ancestral protein, or through convergent evolution, starting from different scaffolds: intriguingly, the active sites of these enzymes are frequently related by a mirror symmetry
Demethylation of vanillic acid by recombinant LigM in a one-pot cofactor regeneration system
No full textIn recent years, by investigating bacterial degradation of lignin, interesting enzymatic activities aimed at
breaking specific linkages were identified. In this work we focused on the tetrahydrofolate (THF)-
dependent O-demethylase LigM from the bacterium Sphingobium sp. strain SYK-6 which converts vanillic
acid to protocatechuic acid (PCA). The recombinant LigM was overexpressed in E. coli up to 80 mg L−1 fermentation
broth, and the main kinetic parameters on the best substrate vanillic acid (kcat = 0.19 s−1 and Km
= 54 μM) and the substrate specificity were identified. The substrate preference was rationalized using a
three-dimensional model of LigM structure in complex with THF, which also allowed us to propose a reaction
mechanism. LigM efficiently converts vanillic acid into PCA but the reaction requires a 10-fold molar
excess of the THF cofactor. In order to limit the cofactor consumption, the plant methionine synthase
MetE enzyme was also overexpressed in E. coli and used in combination with LigM. Under optimized conditions,
the dual enzyme system produced 5 mM PCA using 0.1 mM THF only, a 500-fold decrease in the
cofactor : substrate molar ratio compared to the mono-enzyme process. This represents the first regeneration
method for THF in a biocatalytic process
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