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https://www.readbyqxmd.com/read/29038564/a-quantitative-comparison-of-cytosolic-delivery-via-different-protein-uptake-systems
#1
Wouter P R Verdurmen, Marigona Mazlami, Andreas Plückthun
Over many years, a variety of delivery systems have been investigated that have the capacity to shuttle macromolecular cargoes, especially proteins, into the cytosol. Due to the lack of an objective way to quantify cytosolic delivery, relative delivery efficiencies of the various transport systems have remained unclear. Here, we demonstrate the use of the biotin ligase assay for a quantitative comparison of protein transport to the cytosol via cell-penetrating peptides, supercharged proteins and bacterial toxins in four different cell lines...
October 16, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28972172/a-lower-isoelectric-point-increases-signal-sequence-mediated-secretion-of-recombinant-proteins-through-a-bacterial-abc-transporter
#2
Hyunjong Byun, Jiyeon Park, Sun Chang Kim, Jung Hoon Ahn
Efficient protein production for industrial and academic purposes often involves engineering microorganisms to produce and secrete target proteins into the culture. Pseudomonas fluorescens has a TliDEF ATP-binding cassette transporter, a type I secretion system, which recognizes C-terminal LARD3 signal sequence of thermostable lipase TliA. Many proteins are secreted by TliDEF in vivo when recombined with LARD3, but there are still others that cannot be secreted by TliDEF even when LARD3 is attached. However, the factors that determine whether or not a recombinant protein can be secreted through TliDEF are still unknown...
September 29, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28902449/enzyme-encapsulation-by-a-ferritin-cage
#3
Stephan Tetter, Donald Hilvert
Ferritins, conserved across all kingdoms of life, are protein nanocages that evolved to mineralize iron. The last several decades have shown that these cages have considerable technological and medical potential owing to their stability and tolerance to modification, as well as their ability to template nanoparticle synthesis and incorporate small molecules. Here we show that it is possible to encapsulate proteins in a ferritin cage by exploiting electrostatic interactions with its negatively charged interior...
September 13, 2017: Angewandte Chemie
https://www.readbyqxmd.com/read/28870080/charge-shielding-prevents-aggregation-of-supercharged-gfp-variants-at-high-protein-concentration
#4
Joshua R Laber, Barton J Dear, Matheus L Martins, Devin E Jackson, Andrea DiVenere, Jimmy D Gollihar, Andrew D Ellington, Thomas M Truskett, Keith P Johnston, Jennifer A Maynard
Understanding protein stability is central to combatting protein aggregation diseases and developing new protein therapeutics. At the high concentrations often present in biological systems, purified proteins can exhibit undesirable high solution viscosities and poor solubilities mediated by short-range electrostatic and hydrophobic protein-protein interactions. The interplay between protein amino acid sequence, protein structure, and solvent conditions to minimize protein-protein interactions is key to designing well-behaved pharmaceutical proteins...
October 2, 2017: Molecular Pharmaceutics
https://www.readbyqxmd.com/read/28742233/controlled-supramolecular-self-assembly-of-super-charged-%C3%AE-lactoglobulin-a-peg-conjugates-into-nanocapsules
#5
Amit Kumar Khan, Sushanth Gudlur, Hans-Peter M de Hoog, Winna Siti, Bo Liedberg, Madhavan Nallani
The synthesis and characterization of a new protein-polymer conjugate composed of β lactoglobulin A (βLG A) and poly(ethylene glycol) PEG is described. βLG A was selectively modified to self-assemble by super-charging via amination or succinylation followed by conjugation with PEG. An equimolar mixture of the oppositely charged protein-polymer conjugates self-assemble into spherical capsules of 80-100 nm in diameter. The self-assembly proceeds by taking simultaneous advantage of the amphiphilicity and polyelectrolyte nature of the protein-polymer conjugate...
September 18, 2017: Angewandte Chemie
https://www.readbyqxmd.com/read/28692103/supercharging-spycatcher-toward-an-intrinsically-disordered-protein-with-stimuli-responsive-chemical-reactivity
#6
Yang Cao, Dong Liu, Wen-Bin Zhang
We report a supercharged, intrinsically disordered protein, SpyCatcher(-), possessing stimuli-responsive reactivity toward SpyTag with tunable yields ranging from 4% to 98% depending on pH, temperature, ionic strength, etc. The CD and NMR studies reveal that the reaction occurs through a folded intermediate formed probably via a different mechanism from that of SpyCatcher.
July 10, 2017: Chemical Communications: Chem Comm
https://www.readbyqxmd.com/read/28686014/efficient-dual-sirna-and-drug-delivery-using-engineered-lipoproteoplexes
#7
Che Fu Liu, Raymond Chen, Joseph A Frezzo, Priya Katyal, Lindsay K Hill, Liming Yin, Nikita Srivastava, Haresh T More, P Douglas Renfrew, Richard Bonneau, Jin Kim Montclare
An engineered supercharged coiled-coil protein (CSP) and the cationic transfection reagent Lipofectamine 2000 are combined to form a lipoproteoplex for the purpose of dual delivery of siRNA and doxorubicin. CSP, bearing an external positive charge and axial hydrophobic pore, demonstrates the ability to condense siRNA and encapsulate the small-molecule chemotherapeutic, doxorubicin. The lipoproteoplex demonstrates improved doxorubicin loading relative to Lipofectamine 2000. Furthermore, it induces effective transfection of GAPDH (60% knockdown) in MCF-7 breast cancer cells with efficiencies comparing favorably to Lipofectamine 2000...
August 4, 2017: Biomacromolecules
https://www.readbyqxmd.com/read/28616951/direct-characterization-of-the-native-structure-and-mechanics-of-cyanobacterial-carboxysomes
#8
Matthew Faulkner, Jorge Rodriguez-Ramos, Gregory F Dykes, Siân V Owen, Selene Casella, Deborah M Simpson, Robert J Beynon, Lu-Ning Liu
Carboxysomes are proteinaceous organelles that play essential roles in enhancing carbon fixation in cyanobacteria and some proteobacteria. These self-assembling organelles encapsulate Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase using a protein shell structurally resembling an icosahedral viral capsid. The protein shell serves as a physical barrier to protect enzymes from the cytosol and a selectively permeable membrane to mediate transport of enzyme substrates and products...
August 3, 2017: Nanoscale
https://www.readbyqxmd.com/read/28585364/supercharging-proteins-how-many-charges-can-a-protein-carry
#9
Nicolas C Polfer
Very highly charged proteins, so-called "supercharged" ions, can lose (excess) protons to background gases like N2 . It is remarkable that such extremely acidic species can be generated in electrospray ionization, in the presence of not just N2 but also much higher-basicity solvents. What mechanism(s) can explain such high charging, and what is the ultimate limit?
June 6, 2017: Angewandte Chemie
https://www.readbyqxmd.com/read/28391065/novel-lipoproteoplex-delivers-keap1-sirna-based-gene-therapy-to-accelerate-diabetic-wound-healing
#10
Piul S Rabbani, Anna Zhou, Zachary M Borab, Joseph A Frezzo, Nikita Srivastava, Haresh T More, William J Rifkin, Joshua A David, Samuel J Berens, Raymond Chen, Sophia Hameedi, Muhammad H Junejo, Camille Kim, Rita A Sartor, Che F Liu, Pierre B Saadeh, Jin K Montclare, Daniel J Ceradini
Therapeutics utilizing siRNA are currently limited by the availability of safe and effective delivery systems. Cutaneous diseases, specifically ones with significant genetic components are ideal candidates for topical siRNA based therapy but the anatomical structure of skin presents a considerable hurdle. Here, we optimized a novel liposome and protein hybrid nanoparticle delivery system for the topical treatment of diabetic wounds with severe oxidative stress. We utilized a cationic lipid nanoparticle (CLN) composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the edge activator sodium cholate (NaChol), in a 6:1 ratio of DOTAP:NaChol (DNC)...
July 2017: Biomaterials
https://www.readbyqxmd.com/read/28368095/extended-protein-ions-are-formed-by-the-chain-ejection-model-in-chemical-supercharging-electrospray-ionization
#11
Micah T Donor, Simon A Ewing, Muhammad A Zenaidee, William A Donald, James S Prell
Supercharging electrospray ionization can be a powerful tool for increasing charge states in mass spectra and generating unfolded ion structures, yet key details of its mechanism remain unclear. The structures of highly extended protein ions and the mechanism of supercharging were investigated using ion mobility-mass spectrometry. Head-to-tail-linked polyubiquitins (Ubq1-11) were used to determine size and charge state scaling laws for unfolded protein ions formed by supercharging while eliminating amino acid composition as a potential confounding factor...
April 12, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28281548/structure-and-assembly-of-scalable-porous-protein-cages
#12
Eita Sasaki, Daniel Böhringer, Michiel van de Waterbeemd, Marc Leibundgut, Reinhard Zschoche, Albert J R Heck, Nenad Ban, Donald Hilvert
Proteins that self-assemble into regular shell-like polyhedra are useful, both in nature and in the laboratory, as molecular containers. Here we describe cryo-electron microscopy (EM) structures of two versatile encapsulation systems that exploit engineered electrostatic interactions for cargo loading. We show that increasing the number of negative charges on the lumenal surface of lumazine synthase, a protein that naturally assembles into a ∼1-MDa dodecahedron composed of 12 pentamers, induces stepwise expansion of the native protein shell, giving rise to thermostable ∼3-MDa and ∼6-MDa assemblies containing 180 and 360 subunits, respectively...
March 10, 2017: Nature Communications
https://www.readbyqxmd.com/read/28252115/improving-sensitivity-and-linear-dynamic-range-of-intact-protein-analysis-using-a-robust-and-easy-to-use-microfluidic-device
#13
Gregory T Roman, James P Murphy
We demonstrate an integrated microfluidic LC device coupled to a QTOF capable of improving sensitivity and linearity for intact protein analysis while also tuning the charge state distributions (CSD) of whole antibodies. The mechanism for sensitivity improvement using microflow ESI is demonstrated by shifting of the CSD to higher charge state, and narrowing of the overall CSD. Both of these aspects serve to improve ion current of the most abundant charge state of antibodies and lead to improvement in sensitivity over high flow ESI by a factor of 15×...
March 2, 2017: Analyst
https://www.readbyqxmd.com/read/28187214/what-s-the-remedy-for-the-distal-necrosis-of-diep-flap-better-venous-drain-or-more-arterial-supply
#14
Yi Zhang, Tingliang Wang, Jiao Wei, Jinguang He, Tao Wang, Ying Liu, Hua Xu, Jiasheng Dong
BACKGROUND: We developed a novel pedicled DIEP flap model in rat to explore the possible remedy for the distal necrosis of the flap. METHODS: A deep inferior epigastric perforator (DIEP) flap, based on the second right cranial perforator (P2) as the main pedicle, was elevated in 48 Sprague-Dawley rats. The rats were randomized into 4 groups: group I, the left P2 remaining intact as supercharging; group II, the left P2 artery alone kept as supercharging; group III, the left P2 vein alone kept as supercharging; group IV, no supercharging...
2017: PloS One
https://www.readbyqxmd.com/read/28161552/the-effect-of-charge-mutations-on-the-stability-and-aggregation-of-a-human-single-chain-fv-fragment
#15
James I Austerberry, Rana Dajani, Stanislava Panova, Dorota Roberts, Alexander P Golovanov, Alain Pluen, Christopher F van der Walle, Shahid Uddin, Jim Warwicker, Jeremy P Derrick, Robin Curtis
The aggregation propensities of a series of single-chain variable fragment (scFv) proteins harbouring supercharged sequences, salt bridges and lysine/arginine-enriched motifs were characterised as a function of pH and ionic strength to isolate the electrostatic contributions. Recent improvements in aggregation predictors rely on using knowledge of native-state protein-protein interactions. Consistent with previous findings, electrostatic contributions to native protein-protein interactions correlate with aggregate growth pathway and rates...
February 1, 2017: European Journal of Pharmaceutics and Biopharmaceutics
https://www.readbyqxmd.com/read/27487254/supercharging-reagent-for-enhanced-liquid-chromatographic-separation-and-charging-of-sialylated-and-high-molecular-weight-glycopeptides-for-nanohplc-esi-ms-ms-analysis
#16
Chia-Wei Lin, Micha A Haeuptle, Markus Aebi
Recent developments in proteomic techniques have led to the development of mass spectrometry (MS)-based methods to characterize site-specific glycosylation of proteins. However, appropriate analytical tools to characterize acidic and high-molecular-weight (hMW) glycopeptides are still lacking. In this study, we demonstrate that the addition of supercharging reagent, m-nitrobenzyl alcohol (m-NBA), into mobile phases greatly facilitates the analysis of acidic and hMW glycopeptides. Using commercial glycoproteins, we demonstrated that in the presence of m-NBA the charge state of sialylated glycopeptides increased and the chromatographic separation of neutral and acidic glycopeptides revealed a remarkable improvement...
September 6, 2016: Analytical Chemistry
https://www.readbyqxmd.com/read/27441318/electrothermal-supercharging-of-proteins-in-native-ms-effects-of-protein-isoelectric-point-buffer-and-nanoesi-emitter-tip-size
#17
Daniel N Mortensen, Evan R Williams
The extent of charging resulting from electrothermal supercharging for protein ions formed from various buffered aqueous solutions using nanoESI emitters with tip diameters between ∼1.5 μm and ∼310 nm is compared. Charging increases with decreasing tip size for proteins that are positively charged in solution but not for proteins that are negatively charged in solution. These results suggest that Coulombic attraction between positively charged protein molecules and the negatively charged glass surfaces in the tips of the emitters causes destabilization and even unfolding of proteins prior to nanoESI...
October 7, 2016: Analyst
https://www.readbyqxmd.com/read/27224484/saxs-sans-on-supercharged-proteins-reveals-residue-specific-modifications-of-the-hydration-shell
#18
Henry S Kim, Anne Martel, Eric Girard, Martine Moulin, Michael Härtlein, Dominique Madern, Martin Blackledge, Bruno Franzetti, Frank Gabel
Water molecules in the immediate vicinity of biomacromolecules, including proteins, constitute a hydration layer characterized by physicochemical properties different from those of bulk water and play a vital role in the activity and stability of these structures, as well as in intermolecular interactions. Previous studies using solution scattering, crystallography, and molecular dynamics simulations have provided valuable information about the properties of these hydration shells, including modifications in density and ionic concentration...
May 24, 2016: Biophysical Journal
https://www.readbyqxmd.com/read/27166796/expression-of-functional-human-sialyltransferases-st3gal1-and-st6gal1-in-escherichia-coli
#19
Maria Elena Ortiz-Soto, Jürgen Seibel
Sialyltransferases (STs) are disulfide-containing, type II transmembrane glycoproteins that catalyze the transfer of sialic acid to proteins and lipids and participate in the synthesis of the core structure oligosaccharides of human milk. Sialic acids are found at the outermost position of glycostructures, playing a key role in health and disease. Sialylation is also essential for the production of recombinant therapeutic proteins (RTPs). Despite their importance, availability of sialyltransferases is limited due to the low levels of stable, soluble and active protein produced in bacterial expression systems, which hampers biochemical and structural studies on these enzymes and restricts biotechnological applications...
2016: PloS One
https://www.readbyqxmd.com/read/27093467/mechanism-of-protein-supercharging-by-sulfolane-and-m-nitrobenzyl-alcohol-molecular-dynamics-simulations-of-the-electrospray-process
#20
Haidy Metwally, Robert G McAllister, Vlad Popa, Lars Konermann
Electrospray ionization (ESI) allows the production of intact gas-phase ions from proteins in solution. Nondenaturing solvent conditions usually culminate in low ESI charge states. However, many mass spectrometric applications benefit from protein ions that are more highly charged. One way to boost protein charge is the addition of supercharging agents (SCAs) such as sulfolane or m-nitrobenzyl alcohol (m-NBA) to the aqueous solution. The supercharging mechanism remains controversial. We use molecular dynamics (MD) simulations to examine how SCAs affect the behavior of ESI nanodroplets...
May 17, 2016: Analytical Chemistry
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