Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form

Impact des procédés de transformation sur le devenir digestif des protéines de la viande

In France, meat consumption represents one third of the total protein intake. Meat proteins have a high content of well-balanced essential amino acids near to Human needs. Meat is also a good source of vitamins B12 and iron. Its nutritional potential is thus very higher, but it could be modulated 1) by a protein digestion in the small intestine more or less complete (digestibility), determining the amount of absorbed amino acids, 2) by a speed of digestion higher or lower, affecting the postprandial utilization of absorbed amino acids in protein metabolism. Technological treatments involved in the meat preparation can generate physical-chemical changes of protein. Their impacts on the availability of constitutive amino acids (quantity, kinetics) have been little studied. After determining the impact of unitary operations on meat processing (aging, mincing, cooking) on the biochemical and structural characteristics of proteins, the effect on digestion parameters were studied in vitro on myofibrillar extracts, and in vivo using the minipig as an animal model. The proteomic approach is used in parallel to compare the composition of pig muscles from different rearing methods and study the correlations with digestion. Several protein markers of in vitro digestion are found for both aged and cooked meat, linked to detoxicant process and muscle contraction. The cooking is the process that has the greatest impact on characteristics of "ready-to-eat" product, the application of increasing temperatures (70, 100 and 140°C) resulting in a progressive combination of denaturation, oxidation and then of protein aggregation. Development of an in vitro digestion model (pepsin alone or coupled with trypsin/α-chymotrypsin), on myofibrillar proteins, was used to study the various parameters of digestion (initial slope, maximum rate, time to maximal rate, half-life time and maximal degradation) for the different processes. Aging and mincing have little impacts on these parameters. However, compared to raw meat, the rate of digestion is higher for a cooking temperature to 70°C, which is no longer the case for temperatures above 100°C. Conversely, the potential of maximal degradation is higher for temperatures above 100°C. Linking characteristics of meat with digestion parameters, suggests that for cooking temperatures of around 70°C, denaturation of proteins promotes rapid degradation of proteins by increasing the accessibility of digestive enzymes to their cutting sites, but for higher temperatures, oxidation leads to aggregates that limit accessibility. In vivo study on the minipig, confirms this hypothesis, the rate of appearance of amino acids in the blood following meat ingestion is higher with a core cooking temperature to 75°C, compared to cooking with 60 or 95°C. Furthermore, contrary to what observes in vitro, the use of meat labeled with a stable isotope (15N), we show that the cooking temperature are not affect the total amount of digested proteins in the small intestine (about 95% of ingested proteins). In conclusion, among the unitary operations involved in the preparation of meat, the cooking seems to be the one that most influence the digestion of proteins. The balance between the denaturation processes and protein oxidation according to the cooking temperature appears to be a key point in determining the speed of proteins digestion. This parameter is particularly important in the context of elderly nutrition. As it does not impact on the protein digestibility in the small intestine, the cooking temperature appears as an interesting lever to modulate the speed of meat protein digestion, according to the targeted consumer.Les protéines carnées apportent tous les acides aminés indispensables, en quantité équilibrée par rapport aux besoins de l'Homme. La viande étant également une bonne source de vitamine B12 et de fer, son potentiel nutritionnel est très élevé. Ce potentiel pourrait cependant être modulé 1) par une digestion des protéines plus ou moins complète dans l'intestin grêle, déterminant la quantité d'acides aminés absorbés, 2) par une vitesse de digestion plus ou moins élevée, influençant la capacité d'assimilation des acides aminés absorbés. Les procédés de transformation impliqués dans la préparation des viandes peuvent générer des modifications physicochimiques des protéines. Leurs conséquences sur la mise à disposition des acides aminés constitutifs ont été peu étudiées. Après avoir déterminé l'impact des opérations unitaires de transformation de la viande sur les caractéristiques biochimiques et structurales des protéines, les conséquences sur les paramètres de digestion ont été étudiées, in vitro, sur des extraits myofibrillaires, et in vivo en utilisant le miniporc comme modèle animal. L'approche protéomique est utilisée, en parallèle, pour comparer la composition de muscles de porcs issus de modes d'élevage différents et étudier les corrélations avec la digestion. Plusieurs marqueurs protéiques des paramètres de digestion in vitro sont trouvés sur la viande maturée et cuite, et sont liés au processus de détoxification et de contraction musculaire. La cuisson est le procédé qui a le plus d'impact sur les caractéristiques du produit prêt à consommer, l'application de températures croissantes se traduisant par une combinaison progressive de dénaturation, d'oxydation puis d'agrégation des protéines. Le développement d'un modèle de digestion in vitro, sur des protéines myofibrillaires, a permis d'étudier les différents paramètres de digestion pour les différents procédés. Maturation et hachage ont eu peu d'effets sur ces paramètres. Par contre, par rapport à la viande crue, la vitesse de digestion est supérieure pour une température de cuisson de 70°C, ce qui n'est plus le cas à partir de 100°C. Inversement le potentiel de dégradation maximale est plus élevé pour des températures supérieures à 100°C. La mise en relation des caractéristiques de la viande, avec les paramètres de digestion, suggère que pour des températures de cuisson de l'ordre de 70°C, la dénaturation des protéines favorise une dégradation rapide des protéines en accroissant l'accessibilité des sites de coupure pour les enzymes digestives, mais que pour des températures supérieures, l'oxydation conduit à des agrégats qui limitent cette accessibilité. L'étude in vivo, sur le miniporc, a confirmé cette hypothèse, la vitesse d'apparition des acides aminés dans le sang, suite à l'ingestion des viandes étant supérieure pour une température de cuisson à coeur de 75°C, par rapport à des cuissons à 60 ou 95°C. Par contre contrairement à ce qui était observé in vitro, l'utilisation de viande marquée avec un isotope stable (15N), nous a permis de montrer que la température de cuisson n'affecte pas la quantité totale de protéines digérées dans l'intestin grêle (environ 95% des protéines ingérées). En conclusion, parmi les opérations unitaires impliquées dans la préparation des viandes, la cuisson semble être celle qui influence le plus la digestion des protéines. L'équilibre entre les processus de dénaturation et d'oxydation des protéines en fonction de la température de cuisson semble être un point clé dans la détermination de la vitesse de digestion des protéines. Ce paramètre est important notamment dans le cadre de la nutrition des personnes âgées. Par contre, les compensations au cours des étapes de la digestion font que ces différences de vitesses ne se traduisent pas par des différences de digestibilité dans l'intestin grêle

Impact of technological treatments on digestive future of meat proteins

In France, meat consumption represents one third of the total protein intake. Meat proteins have a high content of well-balanced essential amino acids near to Human needs. Meat is also a good source of vitamins B12 and iron. Its nutritional potential is thus very higher, but it could be modulated 1) by a protein digestion in the small intestine more or less complete (digestibility), determining the amount of absorbed amino acids, 2) by a speed of digestion higher or lower, affecting the postprandial utilization of absorbed amino acids in protein metabolism. Technological treatments involved in the meat preparation can generate physical-chemical changes of protein. Their impacts on the availability of constitutive amino acids (quantity, kinetics) have been little studied. After determining the impact of unitary operations on meat processing (aging, mincing, cooking) on the biochemical and structural characteristics of proteins, the effect on digestion parameters were studied in vitro on myofibrillar extracts, and in vivo using the minipig as an animal model. The proteomic approach is used in parallel to compare the composition of pig muscles from different rearing methods and study the correlations with digestion. Several protein markers of in vitro digestion are found for both aged and cooked meat, linked to detoxicant process and muscle contraction. The cooking is the process that has the greatest impact on characteristics of "ready-to-eat" product, the application of increasing temperatures (70, 100 and 140°C) resulting in a progressive combination of denaturation, oxidation and then of protein aggregation. Development of an in vitro digestion model (pepsin alone or coupled with trypsin/α-chymotrypsin), on myofibrillar proteins, was used to study the various parameters of digestion (initial slope, maximum rate, time to maximal rate, half-life time and maximal degradation) for the different processes. Aging and mincing have little impacts on these parameters. However, compared to raw meat, the rate of digestion is higher for a cooking temperature to 70°C, which is no longer the case for temperatures above 100°C. Conversely, the potential of maximal degradation is higher for temperatures above 100°C. Linking characteristics of meat with digestion parameters, suggests that for cooking temperatures of around 70°C, denaturation of proteins promotes rapid degradation of proteins by increasing the accessibility of digestive enzymes to their cutting sites, but for higher temperatures, oxidation leads to aggregates that limit accessibility. In vivo study on the minipig, confirms this hypothesis, the rate of appearance of amino acids in the blood following meat ingestion is higher with a core cooking temperature to 75°C, compared to cooking with 60 or 95°C. Furthermore, contrary to what observes in vitro, the use of meat labeled with a stable isotope (15N), we show that the cooking temperature are not affect the total amount of digested proteins in the small intestine (about 95% of ingested proteins). In conclusion, among the unitary operations involved in the preparation of meat, the cooking seems to be the one that most influence the digestion of proteins. The balance between the denaturation processes and protein oxidation according to the cooking temperature appears to be a key point in determining the speed of proteins digestion. This parameter is particularly important in the context of elderly nutrition. As it does not impact on the protein digestibility in the small intestine, the cooking temperature appears as an interesting lever to modulate the speed of meat protein digestion, according to the targeted consumer.Les protéines carnées apportent tous les acides aminés indispensables, en quantité équilibrée par rapport aux besoins de l'Homme. La viande étant également une bonne source de vitamine B12 et de fer, son potentiel nutritionnel est très élevé. Ce potentiel pourrait cependant être modulé 1) par une digestion des protéines plus ou moins complète dans l'intestin grêle, déterminant la quantité d'acides aminés absorbés, 2) par une vitesse de digestion plus ou moins élevée, influençant la capacité d'assimilation des acides aminés absorbés. Les procédés de transformation impliqués dans la préparation des viandes peuvent générer des modifications physicochimiques des protéines. Leurs conséquences sur la mise à disposition des acides aminés constitutifs ont été peu étudiées. Après avoir déterminé l'impact des opérations unitaires de transformation de la viande sur les caractéristiques biochimiques et structurales des protéines, les conséquences sur les paramètres de digestion ont été étudiées, in vitro, sur des extraits myofibrillaires, et in vivo en utilisant le miniporc comme modèle animal. L'approche protéomique est utilisée, en parallèle, pour comparer la composition de muscles de porcs issus de modes d'élevage différents et étudier les corrélations avec la digestion. Plusieurs marqueurs protéiques des paramètres de digestion in vitro sont trouvés sur la viande maturée et cuite, et sont liés au processus de détoxification et de contraction musculaire. La cuisson est le procédé qui a le plus d'impact sur les caractéristiques du produit prêt à consommer, l'application de températures croissantes se traduisant par une combinaison progressive de dénaturation, d'oxydation puis d'agrégation des protéines. Le développement d'un modèle de digestion in vitro, sur des protéines myofibrillaires, a permis d'étudier les différents paramètres de digestion pour les différents procédés. Maturation et hachage ont eu peu d'effets sur ces paramètres. Par contre, par rapport à la viande crue, la vitesse de digestion est supérieure pour une température de cuisson de 70°C, ce qui n'est plus le cas à partir de 100°C. Inversement le potentiel de dégradation maximale est plus élevé pour des températures supérieures à 100°C. La mise en relation des caractéristiques de la viande, avec les paramètres de digestion, suggère que pour des températures de cuisson de l'ordre de 70°C, la dénaturation des protéines favorise une dégradation rapide des protéines en accroissant l'accessibilité des sites de coupure pour les enzymes digestives, mais que pour des températures supérieures, l'oxydation conduit à des agrégats qui limitent cette accessibilité. L'étude in vivo, sur le miniporc, a confirmé cette hypothèse, la vitesse d'apparition des acides aminés dans le sang, suite à l'ingestion des viandes étant supérieure pour une température de cuisson à coeur de 75°C, par rapport à des cuissons à 60 ou 95°C. Par contre contrairement à ce qui était observé in vitro, l'utilisation de viande marquée avec un isotope stable (15N), nous a permis de montrer que la température de cuisson n'affecte pas la quantité totale de protéines digérées dans l'intestin grêle (environ 95% des protéines ingérées). En conclusion, parmi les opérations unitaires impliquées dans la préparation des viandes, la cuisson semble être celle qui influence le plus la digestion des protéines. L'équilibre entre les processus de dénaturation et d'oxydation des protéines en fonction de la température de cuisson semble être un point clé dans la détermination de la vitesse de digestion des protéines. Ce paramètre est important notamment dans le cadre de la nutrition des personnes âgées. Par contre, les compensations au cours des étapes de la digestion font que ces différences de vitesses ne se traduisent pas par des différences de digestibilité dans l'intestin grêle

Devenir digestif des matrices animales de type viande

PARTIE III. DES MATRICES COMPLEXES AUX EFFETS MÉTABOLIQUES ET SANTÉ13. Devenir digestif des matrices animales de type viandeDevenir digestif des matrices animales de type viand

Proteomic evolution during steatosis in duck

International audienceThe aim of the study is to better understand the mechanisms underlying the steatosis in birds. Twodimensional electrophoresis gels were performed in liver at different stages of steatosis in duck. Mule ducks were slaughtered after 0, 14 or 23 meals of force-feeding according to commercial conditions. T, Thirty-one proteic spots were differentially expressed between 2 or 3 durations of overfeeding: 3 spots were differentially expressed between the 3 times, 28 between 2 times. The identified proteins (17) could be regrouped into 5 categories: enzymes, traduction factor, proteins involved in cell structure, proteins with antioxidant properties, and proteins that can link calcium

Paramètres de digestion in vitro des protéines carnées

National audienc

Impact of meat cooking and masticatory efficiency on food bolus formation and amino acid absorption in human

International audienceObjectives: Previous studies have shown that both meat cooking conditions, and chewing efficiency, can affect meat protein digestion. The objective of the present study was to try to rank these factors according to the magnitude of their effect, and to evidence potential interactions. Methods: Ten young normo-dented volunteers were invited to participate to the experiment. Pork meat was cooked 10 min at 75°C or 45 min at 90°C, leading to different meat tenderness and cohesiveness. Volunteers were invited to ingest meat in two different situations: after normal mastication, or after deficient mastication (by wearing an occlusal appliance). Meat boluses have been collected before swallowing for assessment of their physical/rheological properties. Plasma aminoacidemia was recorded after ingestion of a meal containing the pork meat as main source of protein. Results: In normal chewing condition and with comparable masticatory durations, the kinetics of matrix disruption was much faster for high cooking meat, and boli were constituted of smaller particles than in low cooking meat. Whatever the meat sample, bolus was softened along the progress of the masticatory sequence and saliva impregnation was similar at the moment of swallowing. By contrast, impaired mastication led to longer and slower sequences, but failed in softening meat samples as well as in reducing it in small fragments. With deficient mastication, the plasma amino acids concentration increases more slowly, and reaches a lower maximal value. This effect was more pronounced for the low cooking meat. Conclusions: Overall, this study showed that a decreased chewing efficiency impairs amino acid absorption, but it also evidenced an interaction between chewing efficiency, the size of swallowed particles (linked to cooking conditions), and the amino acid absorption rate. This work highlights the important but disregarded role of the masticatory function in nutrition, and brings powerful prospects in developing foods for orally impaired people

Protein biomarkers of in vitro digestion parameters of raw and cooked meat

International audienc

Proteomic profile evolution during steatosis development in ducks

International audienceWe investigated a protein profile evolution during steatosis in ducks using 2-dimensional electrophoresis gels to better understand the mechanisms underlying liver steatosis at the level of hepatic proteins in waterfowl. Two-dimensional electrophoresis gels were performed in the liver at different stages of steatosis in the duck. Mule ducks were slaughtered after 0, 14, or 23 meals of overfeeding, according to commercial conditions. Thirty-one proteic spots were differentially expressed between 3 or 2 durations of overfeeding: 3 spots were differentially expressed between the 3 times and 28 spots were differentially expressed between 2 times. The identified proteins (14) could be regrouped into 5 categories: enzymes, translation factors, proteins involved in cell structure, proteins with antioxidant properties, and proteins that can link calcium. This study opens new research areas in the understanding of steatosis in waterfowl, such as cell structure and oxidative stress

La température de cuisson de la viande permet de moduler la vitesse de digestion des protéines, sans affecter la digestibilité dans l’intestin grêle

The nutritional potential of food proteins is modulated by the quantitative digestion in the small intestine (digestibility), which determines the amino acid (AA) bioavailability, and the speed of this digestion, which impacts on AA utilization for body protein synthesis. This study aimed to evaluate the effect of cooking temperature (CT°) on in vivo digestion of meat proteins. Three CT° were applied (30min) to 15N-labelled Longissimus dorsi muscles: 60, 75 and 95°C. Minipigs, fitted with an arterial catheter and an ileal canula, were fed a test meal containing this cooked meat. The speed of digestion, evaluated from the kinetics of indispensable AA appearance in blood, was greater for the CT° of 75°C, compared to the others. The true ileal digestibility, which averaged 95%, was not affected. These results show that CT° can be an interesting lever to modulate the speed of the meat protein digestion, without affecting AA bioavailability