11 results on '"Perry, Sarah"'
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2. Polymer-based microfluidic device for on-chip counter-diffusive crystallization and in situ X-ray crystallography at room temperature.
- Author
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Saha S, Özden C, Samkutty A, Russi S, Cohen A, Stratton MM, and Perry SL
- Subjects
- Crystallography, X-Ray, Crystallization, Temperature, Lab-On-A-Chip Devices, Polymers, Proteins chemistry
- Abstract
Proteins are long chains of amino acid residues that perform a myriad of functions in living organisms, including enzymatic reactions, signalling, and maintaining structural integrity. Protein function is determined directly by the protein structure. X-ray crystallography is the primary technique for determining the 3D structure of proteins, and facilitates understanding the effects of protein structure on function. The first step towards structure determination is crystallizing the protein of interest. We have developed a centrifugally-actuated microfluidic device that incorporates the fluid handling and metering necessary for protein crystallization. Liquid handling takes advantage of surface forces to control fluid flow and enable metering, without the need for any fluidic or pump connections. Our approach requires only the simple steps of pipetting the crystallization reagents into the device followed by either spinning or shaking to set up counter-diffusive protein crystallization trials. The use of thin, UV-curable polymers with a high level of X-ray transparency allows for in situ X-ray crystallography, eliminating the manual handling of fragile protein crystals and streamlining the process of protein structure analysis. We demonstrate the utility of our device using hen egg white lysozyme as a model system, followed by the crystallization and in situ , room temperature structural analysis of the hub domain of calcium-calmodulin dependent kinase II (CaMKIIβ).
- Published
- 2023
- Full Text
- View/download PDF
3. Incorporation of proteins into complex coacervates.
- Author
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Blocher McTigue WC and Perry SL
- Subjects
- Proteins
- Abstract
Complex coacervates have found a renewed interest in the past few decades in various fields such as food and personal care products, membraneless cellular compartments, the origin of life, and, most notably, as a mode of transport and stabilization of drugs. Here, we describe general methods for characterizing the phase behavior of complex coacervates and quantifying the incorporation of proteins into these phase separated materials., (© 2021 Elsevier Inc. All rights reserved.)
- Published
- 2021
- Full Text
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4. How directed evolution reshapes the energy landscape in an enzyme to boost catalysis.
- Author
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Otten R, Pádua RAP, Bunzel HA, Nguyen V, Pitsawong W, Patterson M, Sui S, Perry SL, Cohen AE, Hilvert D, and Kern D
- Subjects
- Catalytic Domain, Nuclear Magnetic Resonance, Biomolecular, Protein Conformation, Biocatalysis, Computer-Aided Design, Directed Molecular Evolution, Enzymes chemistry, Enzymes genetics, Proteins chemistry, Proteins genetics
- Abstract
The advent of biocatalysts designed computationally and optimized by laboratory evolution provides an opportunity to explore molecular strategies for augmenting catalytic function. Applying a suite of nuclear magnetic resonance, crystallography, and stopped-flow techniques to an enzyme designed for an elementary proton transfer reaction, we show how directed evolution gradually altered the conformational ensemble of the protein scaffold to populate a narrow, highly active conformational ensemble and accelerate this transformation by nearly nine orders of magnitude. Mutations acquired during optimization enabled global conformational changes, including high-energy backbone rearrangements, that cooperatively organized the catalytic base and oxyanion stabilizer, thus perfecting transition-state stabilization. The development of protein catalysts for many chemical transformations could be facilitated by explicitly sampling conformational substates during design and specifically stabilizing productive substates over all unproductive conformations., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)
- Published
- 2020
- Full Text
- View/download PDF
5. Protein Encapsulation Using Complex Coacervates: What Nature Has to Teach Us.
- Author
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Blocher McTigue WC and Perry SL
- Subjects
- Protein Stability, Solvents, Proteins chemistry, Water
- Abstract
Protein encapsulation is a growing area of interest, particularly in the fields of food science and medicine. The sequestration of protein cargoes is achieved using a variety of methods, each with benefits and drawbacks. One of the most significant challenges associated with protein encapsulation is achieving high loading while maintaining protein viability. This difficulty is exacerbated because many encapsulant systems require the use of organic solvents. By contrast, nature has optimized strategies to compartmentalize and protect proteins inside the cell-a purely aqueous environment. Although the mechanisms whereby aspects of the cytosol is able to stabilize proteins are unknown, the crowded nature of many newly discovered, liquid phase separated "membraneless organelles" that achieve protein compartmentalization suggests that the material environment surrounding the protein may be critical in determining stability. Here, encapsulation strategies based on liquid-liquid phase separation, and complex coacervation in particular, which has many of the key features of the cytoplasm as a material, are reviewed. The literature on protein encapsulation via coacervation is also reviewed and the parameters relevant to creating protein-containing coacervate formulations are discussed. Additionally, potential opportunities associated with the creation of tailored materials to better facilitate protein encapsulation and stabilization are highlighted., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2020
- Full Text
- View/download PDF
6. Recent Advances in Encapsulation, Protection, and Oral Delivery of Bioactive Proteins and Peptides using Colloidal Systems.
- Author
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Perry SL and McClements DJ
- Subjects
- Administration, Oral, Biopolymers, Chemical Phenomena, Drug Delivery Systems, Drug Liberation, Gastrointestinal Absorption, Humans, Nanoparticles chemistry, Peptides administration & dosage, Peptides pharmacokinetics, Proteins administration & dosage, Proteins pharmacokinetics, Static Electricity, Colloids chemistry, Drug Carriers chemistry, Drug Compounding, Peptides chemistry, Proteins chemistry
- Abstract
There are many areas in medicine and industry where it would be advantageous to orally deliver bioactive proteins and peptides (BPPs), including ACE inhibitors, antimicrobials, antioxidants, hormones, enzymes, and vaccines. A major challenge in this area is that many BPPs degrade during storage of the product or during passage through the human gut, thereby losing their activity. Moreover, many BPPs have undesirable taste profiles (such as bitterness or astringency), which makes them unpleasant to consume. These challenges can often be overcome by encapsulating them within colloidal particles that protect them from any adverse conditions in their environment, but then release them at the desired site-of-action, which may be inside the gut or body. This article begins with a discussion of BPP characteristics and the hurdles involved in their delivery. It then highlights the characteristics of colloidal particles that can be manipulated to create effective BPP-delivery systems, including particle composition, size, and interfacial properties. The factors impacting the functional performance of colloidal delivery systems are then highlighted, including their loading capacity, encapsulation efficiency, protective properties, retention/release properties, and stability. Different kinds of colloidal delivery systems suitable for encapsulation of BPPs are then reviewed, such as microemulsions, emulsions, solid lipid particles, liposomes, and microgels. Finally, some examples of the use of colloidal delivery systems for delivery of specific BPPs are given, including hormones, enzymes, vaccines, antimicrobials, and ACE inhibitors. An emphasis is on the development of food-grade colloidal delivery systems, which could be used in functional or medical food applications. The knowledge presented should facilitate the design of more effective vehicles for the oral delivery of bioactive proteins and peptides.
- Published
- 2020
- Full Text
- View/download PDF
7. Design rules for encapsulating proteins into complex coacervates.
- Author
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Blocher McTigue WC and Perry SL
- Subjects
- Animals, Capsules, Humans, Hydrogen-Ion Concentration, Models, Molecular, Polyglutamic Acid chemistry, Polylysine chemistry, Protein Conformation, Static Electricity, Proteins chemistry
- Abstract
We investigated the encapsulation of the model proteins bovine serum albumin (BSA), human hemoglobin (Hb), and hen egg white lysozyme (HEWL) into two-polymer complex coacervates as a function of polymer and solution conditions. Electrostatic parameters such as pH, protein net charge, salt concentration, and polymer charge density can be used to modulate protein uptake. While the use of a two-polymer coacervation system enables the encapsulation of weakly charged proteins that would otherwise require chemical modification to facilitate electrostatic complexation, we observed significantly higher uptake for proteins whose structure includes a cluster of like-charged residues on the protein surface. In addition to enhancing uptake, the presence of a charge patch also increased the sensitivity of the system to modulation by other parameters, including the length of the complexing polymers. Lastly, our results suggest that the distribution of charge on a protein surface may lead to different scaling behaviour for both the encapsulation efficiency and partition coefficient as a function of the absolute difference between the protein isoelectric point and the solution pH. These results provide insight into possible biophysical mechanisms whereby cells can control the uptake of proteins into coacervate-like granules, and suggest future utility in applications ranging from medicine and sensing to remediation and biocatalysis.
- Published
- 2019
- Full Text
- View/download PDF
8. Complex coacervate-based materials for biomedicine.
- Author
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Blocher WC and Perry SL
- Subjects
- Electrolytes chemistry, Biocompatible Materials, Polymers chemistry, Proteins chemistry
- Abstract
There has been increasing interest in complex coacervates for deriving and transporting biomaterials. Complex coacervates are a dense, polyelectrolyte-rich liquid that results from the electrostatic complexation of oppositely charged macroions. Coacervates have long been used as a strategy for encapsulation, particularly in food and personal care products. More recent efforts have focused on the utility of this class of materials for the encapsulation of small molecules, proteins, RNA, DNA, and other biomaterials for applications ranging from sensing to biomedicine. Furthermore, coacervate-related materials have found utility in other areas of biomedicine, including cartilage mimics, tissue culture scaffolds, and adhesives for wet, biological environments. Here, we discuss the self-assembly of complex coacervate-based materials, current challenges in the intelligent design of these materials, and their utility applications in the broad field of biomedicine. WIREs Nanomed Nanobiotechnol 2017, 9:e1442. doi: 10.1002/wnan.1442 For further resources related to this article, please visit the WIREs website., (© 2016 Wiley Periodicals, Inc.)
- Published
- 2017
- Full Text
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9. Identification and characterization of CRG-L2, a new marker for liver tumor development.
- Author
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Graveel CR, Harkins-Perry SR, Acevedo LG, and Farnham PJ
- Subjects
- Base Sequence, DNA Primers, DNA, Complementary, Humans, In Situ Hybridization, Liver Neoplasms diagnosis, Liver Neoplasms metabolism, Membrane Proteins, Nerve Tissue Proteins, RNA, Messenger genetics, Up-Regulation, Biomarkers, Tumor metabolism, Liver Neoplasms pathology, Proteins metabolism
- Abstract
Liver cancer is very common worldwide and the rates of hepatocellular carcinoma (HCC) have increased by over 70% in the last 2 decades in the US. Late diagnosis, because of the lack of clinical symptoms, and decreased hepatic function, because of underlying hepatic disease, lead to the extremely high mortality rates associated with HCC. Clearly, the identification of markers that are expressed early in the development of HCC and that are easily detected in high-risk patients would aid in early diagnosis and increased survival. We present the cloning and characterization of a novel gene, CRG-L2 (Cancer related gene-Liver 2), which displays high expression in murine and human hepatocellular carcinomas. Using in situ hybridization, we show that CRG-L2 mRNA levels are increased early during the development of liver tumors in C3H/HeJ mice, and that in normal tissues CRG-L2 mRNA is restricted to the murine testis and human placenta. Its restricted expression in normal tissues and unique early upregulation during tumor development make CRG-L2 an excellent candidate as a new clinical marker of HCC.
- Published
- 2003
- Full Text
- View/download PDF
10. Fabrication of X-ray compatible microfluidic platforms for protein crystallization
- Author
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Guha, Sudipto, Perry, Sarah L., Pawate, Ashtamurthy S., and Kenis, Paul J.A.
- Subjects
- *
MICROFABRICATION , *MICROFLUIDIC devices , *CRYSTALLIZATION , *PROTEINS , *THAUMATINS , *CHEMICAL reagents , *X-ray diffraction - Abstract
Abstract: This paper reports a method for fabricating multilayer microfluidic protein crystallization platforms using different materials to achieve X-ray transparency and compatibility with crystallization reagents. To validate this approach, three soluble proteins, lysozyme, thaumatin, and ribonuclease A were crystallized on-chip, followed by on-chip diffraction data collection. We also report a chip with an array of wells for screening different conditions that consume a minimal amount of protein solution as compared to traditional screening methods. A large number of high quality isomorphous protein crystals can be grown in the wells, after which slices of X-ray data can be collected from many crystals still residing within the wells. Complete protein structures can be obtained by merging these slices of data followed by further processing with crystallography software. This approach of using an X-ray transparent chip for screening, crystal growth, and X-ray data collection enables room temperature data collection from many crystals mounted in parallel, which thus eliminates crystal handling and minimizes radiation damage to the crystals. [Copyright &y& Elsevier]
- Published
- 2012
- Full Text
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11. Determination of the Phase Diagram for Soluble and Membrane Proteins.
- Author
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Talreja, Sameer, Perry, Sarah L., Guha, Sudipto, Bhamidi, Venkateswarlu, Zukoski, Charles F., and Kenis, Paul J. A.
- Subjects
- *
MEMBRANE proteins , *PROTEINS - Abstract
Methods to efficiently determine the phase behavior of novel proteins have the potential to significantly benefit structural biology efforts. Here, we present protocols to determine both the solubility boundary and the supersolubility boundary for protein/precipitant systems using an evaporation-based crystallization platform. This strategy takes advantage of the well-defined rates of evaporation that occur in this platform to determine the state of the droplet at any point in time without relying on an equilibrium-based end point. The dynamic nature of this method efficiently traverses phase space along a known path, such that a solubility diagram can be mapped out for both soluble and membrane proteins while using a smaller amount of protein than what is typically used in optimization screens. Furthermore, a variation on this method can be used to decouple crystal nucleation and growth events, so fewer and larger crystals can be obtained within a given droplet. The latter protocol can be used to rescue a crystallization trial where showers of tiny crystals were observed. We validated both of the protocols to determine the phase behavior and the protocol to optimize crystal quality using the soluble proteins lysozyme and ribonuclease A as well as the membrane protein bacteriorhodopsin. [ABSTRACT FROM AUTHOR]
- Published
- 2010
- Full Text
- View/download PDF
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