Structural integrity of protein nanocage at liquid-liquid Interface

Globular proteins adsorb at the interface of two immiscible liquids by maintaining thermodynamically favorable state which often results in a denatured structure and compromised functionalities. However, the behavior of highly structural proteins at the interface of two immiscible liquids is still unexplored. In this study, we focused on the structural behavior of supramolecular protein at the interface. Our previous studies show that highly structural protein adsorbs at the interface and act as a Pickering emulsifier. Theoretical analyses by Molecular Dynamic Simulation proved that the supramolecular protein E2, a highly structured protein nanocage, has retained structural integrity at the liquid-liquid interface. Further, experimental analyses by Small angle X-ray scattering (SAXS) and quartz crystal microbalance and dissipation (QCM-D) confirm the adsorption of E2 on the liquid-liquid interface with zero penetration depth. Moreover, molecular structural analyses using Circular Dichroism (CD) and tryptophan fluorescence for secondary and tertiary structures respectively, also suggest the structural integrity of the cage structure of E2 at the oil-water interface. This study brings new insights into the behavior of highly symmetrical supramolecular protein assembly at the liquid-liquid interface

Protein nanocages for cutaneous delivery

The skin protects the body from UV-induced DNA damage by the sun exposure through the pigment, melanin produced by the melanocytes. This pigment is sometimes over-expressed leading to pigmentation disorders such as melasma. Current treatment involves using tyrosinase inhibitors and lasers, leads to complications such as depigmentation, irritation, and dermatitis, with only 50% patient response. This is mainly due the inability of the delivery system to penetrate the stratum corneum layer of the skin and its non-specificity to the melanocytes. This project is aimed at engineering E2 protein nanocage for enhanced penetration into the stratum corneum layer of the epidermis and targeting/penetrating the melanocytes for the delivery of therapeutics. Genetic fusion of SPACE (Skin Penetrating And Cell Entering) peptide to the E2 nanocage helps its transduction through the stratum corneum layer, in vivo and to the interior of the melanocytes in vitro. Further modification of the E2 protein cage with targeting ligands can facilitate its uptake in melanocytes through the corresponding cell membrane receptors. Multiple modifications could also be imparted to the E2 protein cages without affecting its self-assembly, thereby aiding both penetration and targeting functions for drug delivery. Successful delivery of the engineered protein cages can aid the formulation of novel protein-based drug releasing molecules to be applied to the skin, which can be biocompatible with efficient pharmacokinetics

Protein nanocages as novel biosurfactant in the formulation of Pickering emulsion and gel

Protein nanocages have been shown to be versatile for multitude of applications in biomedicine [1]. Our group has recently reported that the self-assembling protein nanocages localize at oil-water interface and stabilize 200-400 nm nanoemulsions [2]. The protein nanocages are produced using microbial fermentation and purified using conventional chromatography technique. The protein nanocage-stabilized Pickering emulsion are produced by facile sonication technique. The emulsion has been shown to be pH responsive when the pH is switched between 4 and 8. The switch is reversible up to 6 times. The emulsion is stable for more than 2 years. Varying the mass fraction of the protein nanocages/oil results in a shift in rheology from emulsion to gel. The unique properties of the protein nanocages emulsions have attracted industrial interests and we are currently working with our industry collaborators to encapsulate their cosmetic ingredients. We have shown the potential of protein nanocages as a novel biosurfactant that are of interests to the cosmetic industry. Please click Additional Files below to see the full abstract

MEDICINAL FORMULATIONS OF A KANDA TRIBAL HEALER – A TRIBE ON THE VERGE OF DISAPPEARANCE IN BANGLADESH

The Kanda tribe is one of the lesser known small tribes of Bangladesh with an estimated population of about 1700 people (according to them), and on the verge of extinction as a separate entity. To some extent, they have assimilated with the surrounding mainstream Bengali-speaking population, but they still maintain their cultural practices including traditional medicinal practices, for which they have their own tribal healers. Nothing at all has been documented thus far about their traditional medicinal practices and formulations, which are on the verge of disappearance. The Kanda tribe can be found only in scattered tea gardens of Sreemangal in Sylhet district of Bangladesh; dispersion of the tribe into small separated communities is also contributing to the fast losing of traditional medicinal practices. The objective of the present study was to conduct an ethnomedicinal survey among the traditional healers of the Kanda tribe (in fact, only one such healer was found after extensive searches). Information was collected from the healer with the help of a semi-structured questionnaire and the guided field-walk method. A total of 24 formulations were obtained from the healer containing 34 plants including two plants, which could not be identified. Besides medicinal plants, the Kanda healer also used the body hairs of the Asiatic black bear (Ursus thibetanus) and bats (Pteropus giganteus giganteus) in one of his formulation for treatment of fever with shivering. The ailments treated by the Kanda healer were fairly common ailments like cuts and wounds, skin diseases, helminthiasis, fever, respiratory problems (coughs, asthma), gastrointestinal disorders (stomach pain, constipation, diarrhea), burning sensations during urination, various types of pain (headache, body ache, toothache, ear ache), conjunctivitis, poisonous snake, insect or reptile bites, jaundice, and bone fractures. A number of important drugs in allopathic medicine like quinine, artemisinin, and morphine (to name only a few) have been discovered from observing indigenous medicinal practices. From that view point, the formulations used by the Kanda healer merit scientific studies for their potential in the discovery of cheap and effective new drugs. Scientific validation of the medicinal formulations of the Kanda healer can also be effective for treatment of ailments among this tribe, which does not have or does not want to have any contact with modern medicine

E2 protein nanocage as a pickering emulsifier and its applications

Particle-stabilized emulsion or Pickering emulsion has drawn attention in recent years because of its several attractive features. Pickering emulsion offers higher stability in different conditions and it has found applications in a variety of industries including food, pharmaceuticals, and consumer products. The selection of particles or Pickering emulsifier is crucial to engineering the characteristic features of Pickering emulsion. Inorganic, organic as well as hybrid nanoparticles have been used in stabilizing Pickering emulsions for different applications with some identified limitations. Inorganic particle is less preferred for the development of food and pharmaceutical products due to its limited biocompatibility. Organic particles have low stability in different conditions presenting a research gap and a quest for new organic materials to overcome these issues. In this work, a protein-based material, E2 protein nanocage, has been introduced as a new Pickering emulsifier. E2 is the first non-viral protein nanocage to be used in stabilizing Pickering emulsion. E2 exhibits excellent surface activity because of the amphiphilic nature of its external surface. E2 adsorbs at the liquid-liquid interface and provides kinetic stability to the emulsion. The E2-stabilized emulsion exhibits pH-switchability for at least 5 cycles and shows stability in a wide range of pH (neutral to basic), ionic strength (as high as 250 mM NaCl) and storage temperature (up to 50°C). The increase in oil fraction in the emulsion composition results in the formation of gel-like structure. The rheological analysis of Pickering emulsion and gel-like structure confirmed the higher mechanical strength, long-term stability and elasticity of the gel-like structure compared to the emulsion. The structural integrity of E2 at the oil/water interface has been studied experimentally and theoretically. The adsorption kinetics of E2 on model hydrophobic surface confirms the formation of 26 nm in a thick monolayer of protein nanocage. In-depth study of the molecular structure reveals an intact tertiary structure of E2 at the interface while the secondary structure results in an increase in alpha-helicity. The trends of denaturation of E2 protein in different denaturation conditions confirm the trend of structural change of E2 at the interface. Furthermore, the theoretical analysis suggests that hydrophobic force dominates over surface force indicating the structural integrity of protein cage at the interface. To explore a potential application in formulating functional food products, of E2-stabilized Pickering emulsion has been proposed as a platform to deliver active molecules in the gastrointestinal tract with two-step release mechanism. Micronutrients such as iron or calcium can be loaded in the E2 while lipophilic micronutrients in the oil phase of the emulsion. The digestion profile of E2 and E2-stabilized emulsion has been investigated in-vitro using gastric simulated fluid followed by the analysis of the micronutrients bioaccessibility in the first step in stomach phase. In summary, E2 has been shown to stabilize emulsion with pH-responsive behavior. Examination of its structure suggests that it maintains a cage-like structure at the oil/water interface. The non-viral origin combined with the loading of active molecules in both the E2 interior and the oil phase makes E2-stabilized Pickering emulsion attractive as nanocarriers with two-step release mechanism for functional food formulation.Doctor of Philosophy (SCBE

Protein Nanocage as a pH-Switchable Pickering Emulsifier

Encapsulation of active compounds in Pickering emulsions using bioderived protein-based stabilizers holds potential for the development of novel formulations in the fields of foods and cosmetics. We employ a dodecahedron hollow protein nanocage as a pH-switchable Pickering emulsifier. E2 protein nanocages are derived from pyruvate dehydrogenase multienzyme complex from <i>Geobacillus stearothermophilus</i> which adsorb at the oil/water interface at neutral and basic pH’s and stabilize the Pickering emulsions, while in the acidic range, at pH ∼4, the emulsion separates into emulsion and serum phases due to flocculation. The observed process is reversible for at least five cycles. Optimal formulation of a Pickering emulsion composed of rosemary oil, an essential oil, and water has been achieved by ultrasonication and results in droplets of approximately 300 nm in diameter with an oil/water ratio of 0.11 (v/v) and 0.30–0.35% (wt %). Ionic stabilization is observed for concentrations up to 250 mM NaCl and pH values from 7 to 11. The emulsions are stable for at least 10 days when stored at different temperatures up to 50 °C. The resulting Pickering emulsions of different compositions also form a gel-like structure and show shear thinning behavior under shear stress at a higher oil/water ratio

Holistic engineering of cell-free systems through proteome-reprogramming synthetic circuits.

Synthetic biology has focused on engineering genetic modules that operate orthogonally from the host cells. A synthetic biological module, however, can be designed to reprogram the host proteome, which in turn enhances the function of the synthetic module. Here, we apply this holistic synthetic biology concept to the engineering of cell-free systems by exploiting the crosstalk between metabolic networks in cells, leading to a protein environment more favorable for protein synthesis. Specifically, we show that local modules expressing translation machinery can reprogram the bacterial proteome, changing the expression levels of more than 700 proteins. The resultant feedback generates a cell-free system that can synthesize fluorescent reporters, protein nanocages, and the gene-editing nuclease Cas9, with up to 5-fold higher expression level than classical cell-free systems. Our work demonstrates a holistic approach that integrates synthetic and systems biology concepts to achieve outcomes not possible by only local, orthogonal circuits

Holistic engineering of cell-free systems through proteome-reprogramming synthetic circuits.

Synthetic biology has focused on engineering genetic modules that operate orthogonally from the host cells. A synthetic biological module, however, can be designed to reprogram the host proteome, which in turn enhances the function of the synthetic module. Here, we apply this holistic synthetic biology concept to the engineering of cell-free systems by exploiting the crosstalk between metabolic networks in cells, leading to a protein environment more favorable for protein synthesis. Specifically, we show that local modules expressing translation machinery can reprogram the bacterial proteome, changing the expression levels of more than 700 proteins. The resultant feedback generates a cell-free system that can synthesize fluorescent reporters, protein nanocages, and the gene-editing nuclease Cas9, with up to 5-fold higher expression level than classical cell-free systems. Our work demonstrates a holistic approach that integrates synthetic and systems biology concepts to achieve outcomes not possible by only local, orthogonal circuits

Holistic engineering of cell-free systems through proteome-reprogramming synthetic circuits

Synthetic biological modules can be used to reprogram host proteomes, which in turn enhance the function of the synthetic modules. The authors use this holistic synthetic biology approach to engineer a more favorable environment for cell-free protein synthesis