基礎講座 電気泳動(Electrophoresis)

Irreversible protein aggregation is problematic in the biotechnology industry, where aggregation is encountered throughout the lifetime of a therapeutic protein, including during refolding, purification, sterilization, shipping, and storage processes. The purpose of the current review is to provide a fundamental understanding of the mechanisms by which proteins aggregate and by which varying solution conditions, such as temperature, pH, salt type, salt concentration, cosolutes, preservatives, and surfactants, affect this process.

Physical Stability of Proteins in Aqueous Solution: Mechanism and Driving Forces in Nonnative Protein Aggregation

The spectrin-based membrane skeleton of the humble mammalian erythrocyte has provided biologists with a set of interacting proteins with diverse roles in organization and survival of cells in metazoan organisms. This review deals with the molecular physiology of spectrin, ankyrin, which links spectrin to the anion exchanger, and two spectrin-associated proteins that promote spectrin interactions with actin: adducin and protein 4.1. The lack of essential functions for these proteins in generic cells grown in culture and the absence of their genes in the yeast genome have, until recently, limited advances in understanding their roles outside of erythrocytes. However, completion of the genomes of simple metazoans and application of homologous recombination in mice now are providing the first glimpses of the full scope of physiological roles for spectrin, ankyrin, and their associated proteins. These functions now include targeting of ion channels and cell adhesion molecules to specialized compartments within the plasma membrane and endoplasmic reticulum of striated muscle and the nervous system, mechanical stabilization at the tissue level based on transcellular protein assemblies, participation in epithelial morphogenesis, and orientation of mitotic spindles in asymmetric cell divisions. These studies, in addition to stretching the erythrocyte paradigm beyond recognition, also are revealing novel cellular pathways essential for metazoan life. Examples are ankyrin-dependent targeting of proteins to excitable membrane domains in the plasma membrane and the Ca(2+) homeostasis compartment of the endoplasmic reticulum. Exciting questions for the future relate to the molecular basis for these pathways and their roles in a clinical context, either as the basis for disease or more positively as therapeutic targets.

Spectrin and Ankyrin-Based Pathways: Metazoan Inventions for Integrating Cells Into Tissues

Protein aggregation correlates with the development of several debilitating human disorders of growing incidence, such as Alzheimer's and Parkinson's diseases. On the biotechnological side, protein production is often hampered by the accumulation of recombinant proteins into aggregates. Thus, the development of methods to anticipate the aggregation properties of polypeptides is receiving increasing attention. AGGRESCAN is a web-based software for the prediction of aggregation-prone segments in protein sequences, the analysis of the effect of mutations on protein aggregation propensities and the comparison of the aggregation properties of different proteins or protein sets. AGGRESCAN is based on an aggregation-propensity scale for natural amino acids derived from in vivo experiments and on the assumption that short and specific sequence stretches modulate protein aggregation. The algorithm is shown to identify a series of protein fragments involved in the aggregation of disease-related proteins and to predict the effect of genetic mutations on their deposition propensities. It also provides new insights into the differential aggregation properties displayed by globular proteins, natively unfolded polypeptides, amyloidogenic proteins and proteins found in bacterial inclusion bodies. By identifying aggregation-prone segments in proteins, AGGRESCAN http://bioinf.uab.es/aggrescan/
 shall facilitate (i) the identification of possible therapeutic targets for anti-depositional strategies in conformational diseases and (ii) the anticipation of aggregation phenomena during storage or recombinant production of bioactive polypeptides or polypeptide sets.

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AGGRESCAN: a server for the prediction and evaluation of "hot spots" of aggregation in polypeptides

The principles of isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) are reviewed together with the basic thermodynamic formalism on which the two techniques are based. Although ITC is particularly suitable to follow the energetics of an association reaction between biomolecules, the combination of ITC and DSC provides a more comprehensive description of the thermodynamics of an associating system. The reason is that the parameters DeltaG, DeltaH, DeltaS, and DeltaCp obtained from ITC are global properties of the system under study. They may be composed to varying degrees of contributions from the binding reaction proper, from conformational changes of the component molecules during association, and from changes in molecule/solvent interactions and in the state of protonation.

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Isothermal titration calorimetry and differential scanning calorimetry as complementary tools to investigate the energetics of biomolecular recognition.

Amyloid aggregation is a hallmark in many neuropathologies and other diseases of tremendous impact. It is increasingly evident that neuronal death associated with Alzheimer's disease (AD) is mainly produced by oligomers of the amyloid-β (Aβ) peptide. Yet little is known about the detailed structural and biophysical mechanisms of their formation. This lack of complete understanding comes from the labile nature and handling complexity of the oligomers. Consequently, providing reproducible and robust protocols for oligomer preparation is of particular importance.In this study, we describe detailed methods for the preparation and isolation of micellar oligomers of Aβ that evolve towards larger and more stable oligomers enriched in beta-sheet structure and able to acquire a higher capacity to fibrillate. We also describe briefly some biophysical experiments allowing oligomer characterization. 

Preparation and Investigation of Crucial Oligomers in the Early Stages of Aβ40 and Aβ42 Aggregation.

The disclosure relates to immunological compositions for vaccinating human beings against infection by the Human Immunodeficiency Virus (HIV).

Immunological compositions against hiv

Since the beginning of the COVID-19 pandemic, considerable efforts have been made to develop protective vaccines against SARS-CoV-2 infection. However, immunity tends to decline within a few months, and new virus variants are emerging with increased transmissibility and capacity to evade natural or vaccine-acquired immunity. Therefore, new robust strategies are needed to combat SARS-CoV-2 infection. The viral spike composed of S1 and S2 subunits mediates viral attachment and membrane fusion to infect the host cell. In this process, interaction between the highly conserved heptad repeat 1 and 2 regions (HR1 and HR2) of S2 is crucial and for this reason; these regions are promising targets to fight SARS-CoV-2. Here, we describe the design and characterization of chimeric proteins that structurally imitate the S2 HR1 region in a trimeric coiled-coil conformation. We biophysically characterized the proteins and determined their capacity to bind the HR2 region, as well as their inhibitory activity of SARS-CoV-2 infection in vitro. HR1 mimetic proteins showed conformational heterogeneity and a propensity to form oligomers. Moreover, their structure is composed of subdomains with varied stability. Interestingly, the full HR1 proteins showed high affinity for HR2-derived peptides and SARS-CoV-2 inhibitory activity, whereas smaller proteins mimicking HR1 subdomains had a decreased affinity for their complementary HR2 region and did not inhibit the virus. The results provide insight into effective strategies to create mimetic proteins with broad inhibitory activity and therapeutic potential against SARS-CoV-2.

/pdf/exploring-highly-conserved-regions-of-sars-cov-2-spike-s2-3dqj02wv.pdf

Exploring Highly Conserved Regions of SARS-CoV-2 Spike S2 Subunit as Targets for Fusion Inhibition Using Chimeric Proteins

The disclosure relates to immunological compositions for vaccinating human beings against infection by the Human Immunodeficiency Virus (HIV)

Francisco Conejero-Lara

Papers

Preparation and Investigation of Crucial Oligomers in the Early Stages of Aβ40 and Aβ42 Aggregation.

Immunological compositions against hiv

Exploring Highly Conserved Regions of SARS-CoV-2 Spike S2 Subunit as Targets for Fusion Inhibition Using Chimeric Proteins

Immunological compositions comprising hiv gp41 polypeptide derivatives