Chemical modification of edible sodium caseinate: A new grafting method of oleic acid. Characterization and thermal properties of the conjugate.

Sodium caseinate is a well-known amphiphilic protein derived from natural products currently used for the preparation of edible films. To improve some properties, especially to decrease the hydrophilicity and water solubility of the caseinate, the covalent grafting of a hydrophobic edible fatty acid, namely oleic acid, onto caseinate, appears to be a solution. We describe a new synthesis method for the chemical modification of sodium caseinate involving the synthesis of an acid chloride derivative from oleic acid and a phase transfer catalysis reaction in a biphasic medium. Under these conditions, free amine and alcohol groups of the caseinate are likely to be grafted with a fairly high (>50 %) substitution degree. The caseinate derivative is finely characterized, in particular by DOSY NMR, to assess the formation of a casein/oleic acid grafted compound as well as the absence of residual oleic acid.

Structural nanotechnology: three-dimensional cryo-EM and its use in the development of nanoplatforms for in vitro catalysis.

The organization of enzymes into different subcellular compartments is essential for correct cell function. Protein-based cages are a relatively recently discovered subclass of structurally dynamic cellular compartments that can be mimicked in the laboratory to encapsulate enzymes. These synthetic structures can then be used to improve our understanding of natural protein-based cages, or as nanoreactors in industrial catalysis, metabolic engineering, and medicine. Since the function of natural protein-based cages is related to their three-dimensional structure, it is important to determine this at the highest possible resolution if viable nanoreactors are to be engineered. Cryo-electron microscopy (cryo-EM) is ideal for undertaking such analyses within a feasible time frame and at near-native conditions. This review describes how three-dimensional cryo-EM is used in this field and discusses its advantages. An overview is also given of the nanoreactors produced so far, their structure, function, and applications.

Protecting Encapsulin Nanoparticles with Cysteine-Knot Miniproteins.

A big hurdle for the use of protein-based drugs is that they are easily degraded by proteases in the human body. In an attempt to solve this problem, we show the possibility to functionalize TM encapsulin nanoparticles with an mEETI-II knottin miniprotein from the cysteine-stabilized knot class. The resulting particles did not show aggregation and retained part of their protease inhibitive function. This imposes a protection toward protease, in this case, trypsin, degradation of the protein cage. The used chemistry is easy to apply and thus suitable to protect other protein systems from degradation. In addition, this proof of principle opens up the use of other knottins or cysteine-stabilized knots, which can be attached to protein cages to create a heterofunctionalized protein nanocage. This allows specific targeting and tumor suppression among other types of functionalization. Overall, this is a promising strategy to protect a protein of interest which brings oral administration of protein-based drugs one step closer.

Structural Characterization of Native and Modified Encapsulins as Nanoplatforms for in Vitro Catalysis and Cellular Uptake.

Recent years have witnessed the emergence of bacterial semiorganelle encapsulins as promising platforms for bio-nanotechnology. To advance the development of encapsulins as nanoplatforms, a functional and structural basis of these assemblies is required. Encapsulin from Brevibacterium linens is known to be a protein-based vessel for an enzyme cargo in its cavity, which could be replaced with a foreign cargo, resulting in a modified encapsulin. Here, we characterize the native structure of B. linens encapsulins with both native and foreign cargo using cryo-electron microscopy (cryo-EM). Furthermore, by harnessing the confined enzyme (i.e., a peroxidase), we demonstrate the functionality of the encapsulin for an in vitro surface-immobilized catalysis in a cascade pathway with an additional enzyme, glucose oxidase. We also demonstrate the in vivo functionality of the encapsulin for cellular uptake using mammalian macrophages. Unraveling both the structure and functionality of the encapsulins allows transforming biological nanocompartments into functional systems.

Triethylenetetramine prevents insulin aggregation and fragmentation during copper catalyzed oxidation.

Metal catalyzed oxidation via the oxidative system Cu(2+)/ascorbate is known to induce aggregation of therapeutic proteins, resulting in enhanced immunogenicity. Hence, inclusion of antioxidants in protein formulations is of great interest. In this study, using recombinant human insulin (insulin) as a model, we investigated the ability of several excipients, in particular triethylenetetramine (TETA), reduced glutathione(GSH) and ethylenediamine tetraacetic acid (EDTA), for their ability to prevent protein oxidation, aggregation, and fragmentation. Insulin (1mg/ml) was oxidized with 40 µM Cu(2+) and 4mM ascorbic acid in absence or presence of excipients. Among the excipients studied, 1mM of TETA, EDTA, or GSH prevented insulin aggregation upon metal catalyzed oxidation (MCO) for 3h at room temperature, based on size exclusion chromatography (SEC). At lower concentration (100 µM), for 72 h at +4 °C, TETA was the only one to inhibit almost completely oxidation-induced insulin aggregation, fragmentation, and structural changes, as indicated by SEC, nanoparticle tracking analysis, light obscuration particle counting, intrinsic/extrinsic fluorescence, circular dichroism, and chemical derivatization. In contrast, GSH had a slight pro-oxidant effect, as demonstrated by the higher percentage of aggregates and a more severe structural damage, whereas EDTA offered substantially less protection. TETA also protected a monoclonal IgG1 against MCO-induced aggregation, suggesting its general applicability. In conclusion, TETA is a potential candidate excipient for inclusion in formulations of oxidation-sensitive proteins.