11 research outputs found
In-house preparation of hydrogels for batch affinity purification of glutathione <it>S</it>-transferase tagged recombinant proteins
<p>Abstract</p> <p>Background</p> <p>Many branches of biomedical research find use for pure recombinant proteins for direct application or to study other molecules and pathways. Glutathione affinity purification is commonly used to isolate and purify glutathione S-transferase (GST)-tagged fusion proteins from total cellular proteins in lysates. Although GST affinity materials are commercially available as glutathione immobilized on beaded agarose resins, few simple options for in-house production of those systems exist. Herein, we describe a novel method for the purification of GST-tagged recombinant proteins.</p> <p>Results</p> <p>Glutathione was conjugated to low molecular weight poly(ethylene glycol) diacrylate (PEGDA) via thiol-ene “click” chemistry. With our in-house prepared PEGDA:glutathione (PEGDA:GSH) homogenates, we were able to purify a glutathione S-transferase (GST) green fluorescent protein (GFP) fusion protein (GST-GFP) from the soluble fraction of <it>E. coli</it> lysate. Further, microspheres were formed from the PEGDA:GSH hydrogels and improved protein binding to a level comparable to purchased GSH-agarose beads.</p> <p>Conclusions</p> <p>GSH containing polymers might find use as in-house methods of protein purification. They exhibited similar ability to purify GST tagged proteins as purchased GSH agarose beads.</p
Reducible Micelleplexes are Stable Systems for Anti-miRNA Delivery in Cerebrospinal Fluid
Glioblastoma
multiforme (GBM) and other central nervous system
(CNS) cancers have poor long-term prognosis, and there is a significant
need for improved treatments. GBM initiation and progression are mediated,
in part, by microRNA (miRNA), which are endogenous posttranscriptional
gene regulators. Misregulation of miRNAs is a potential target for
therapeutic intervention in GBM. In this work, a micelle-like nanoparticle
delivery system based upon the block copolymer polyÂ(ethylene glycol-<i>b</i>-lactide-<i>b</i>-arginine) was designed with
and without a reducible linkage between the lactide and RNA-binding
peptide, R<sub>15</sub>, to assess the ability of the micelle-like
particles to disassemble. Using confocal live cell imaging, intracellular
dissociation was pronounced for the reducible micelleplexes. This
dissociation was also supported by higher efficiency in a dual luciferase
assay specific for the miRNA of interest, miR-21. Notably, micelleplexes
were found to have significantly better stability and higher anti-miRNA
activity in cerebrospinal fluid than in human plasma, suggesting an
advantage for applying micelleplexes to CNS diseases and in vivo CNS
therapeutics. The reducible delivery system was determined to be a
promising delivery platform for the treatment of CNS diseases with
miRNA therapy
High and Low Molecular Weight Hyaluronic Acid Differentially Influence Macrophage Activation
Macrophages exhibit phenotypic diversity
permitting wide-ranging
roles in maintaining physiologic homeostasis. Hyaluronic acid, a major
glycosaminoglycan of the extracellular matrix, has been shown to have
differential signaling based on its molecular weight. With this in
mind, the main objective of this study was to elucidate the role of
hyaluronic acid molecular weight on macrophage activation and reprogramming.
Changes in macrophage activation were assessed by activation state
selective marker measurement, specifically quantitative real time
polymerase chain reaction, and cytokine enzyme-linked immunoassays,
after macrophage treatment with differing molecular weights of hyaluronic
acid under four conditions: the resting state, concurrent with classical
activation, and following inflammation involving either classically
or alternatively activated macrophages. Regardless of initial polarization
state, low molecular weight hyaluronic acid induced a classically
activated-like state, confirmed by up-regulation of pro-inflammatory
genes, including <i>nos2</i>, <i>tnf</i>, <i>il12b</i>, and <i>cd80,</i> and enhanced secretion
of nitric oxide and TNF-α. High molecular weight hyaluronic
acid promoted an alternatively activated-like state, confirmed by
up regulation of pro-resolving gene transcription, including <i>arg1</i>, <i>il10</i>, and <i>mrc1,</i> and
enhanced arginase activity. Overall, our observations suggest that
macrophages undergo phenotypic changes dependent on molecular weight
of hyaluronan that correspond to either (1) pro-inflammatory response
for low molecular weight HA or (2) pro-resolving response for high
molecular weight HA. These observations bring significant further
understanding of the influence of extracellular matrix polymers, hyaluronic
acid in particular, on regulating the inflammatory response of macrophages.
This knowledge can be used to guide the design of HA-containing biomaterials
to better utilize the natural response to HAs
Combination of Oncolytic Herpes Simplex Viruses Armed with Angiostatin and IL-12 Enhances Antitumor Efficacy in Human Glioblastoma Models
Oncolytic herpes simplex virus (oHSV) can potentially spread throughout the tumor, reach isolated infiltrating cells, kill them, and deliver anticancer agents. However, the host responds to oHSV by inducing intratumoral infiltration of macrophages that can engulf the virus, limiting the potential of this therapeutic strategy. Hypervascularity is a pathognomonic feature of glioblastoma (GBM) and is a promising therapeutic target. Antiangiogenic treatments have multiple benefits, including the capacity to increase oHSV efficacy by suppressing macrophage extravasation and infiltration into the tumor. Angiostatin is an antiangiogenic polypeptide, and interleukin-12 (IL-12) is an immunostimulatory cytokine with strong antiangiogenic effects. Clinical use of each has been limited by delivery issues and systemic toxicity.We tested a combination treatment strategy using oHSVs expressing angiostatin (G47Δ-mAngio) and IL-12 (G47Δ-mIL12) in two orthotopic human GBMmodels. Intratumoral injection of G47Δ-mAngio and G47Δ-mIL12 in mice bearing intracranial U87 or tumors derived from glioblastoma stem cells significantly prolonged survival compared to each armed oHSV alone. This was associated with increased antiangiogenesis and virus spread and decreased macrophages. These data support the paradigm of using oHSV expressing different antiangiogenic agents and show for the first time that oHSVs expressing angiostatin and IL-12 can improve efficacy in human GBM models
Allosteric Modulation of Ras-GTP Is Linked to Signal Transduction through RAF Kinase*
Ras is a key signal transduction protein in the cell. Mutants of Gly12 and Gln61 impair GTPase activity and are found prominently in cancers. In wild type Ras-GTP, an allosteric switch promotes disorder to order transition in switch II, placing Gln61 in the active site. We show that the “on” and “off” conformations of the allosteric switch can also be attained in RasG12V and RasQ61L. Although both mutants have similarly impaired active sites in the on state, RasQ61L stabilizes an anti-catalytic conformation of switch II in the off state of the allosteric switch when bound to Raf. This translates into more potent activation of the MAPK pathway involving Ras, Raf kinase, MEK, and ERK (Ras/Raf/MEK/ERK) in cells transfected with RasQ61L relative to RasG12V. This differential is not observed in the Raf-independent pathway involving Ras, phosphoinositide 3-kinase (PI3K), and Akt (Ras/PI3K/Akt). Using a combination of structural analysis, hydrolysis rates, and experiments in NIH-3T3 cells, we link the allosteric switch to the control of signaling in the Ras/Raf/MEK/ERK pathway, supporting a GTPase-activating protein-independent model for duration of the Ras-Raf complex
Distinguishing Inflammation from Tumor and Peritumoral Edema by Myeloperoxidase Magnetic Resonance Imaging
Proteolytically activated anti-bacterial hydrogel microspheres
Hydrogels are finding increased clinical utility as advances continue to exploit their favorable material properties. Hydrogels can be adapted for many applications, including surface coatings and drug delivery. Anti-infectious surfaces and delivery systems that actively destroy invading organisms are alternative ways to exploit the favorable material properties offered by hydrogels. Sterilization techniques are commonly employed to ensure the materials are non-infectious upon placement, but sterilization is not absolute and infections are still expected. Natural, anti-bacterial proteins have been discovered which have the potential to act as anti-infectious agents; however, the proteins are toxic and need localized release to have therapeutic efficacy without toxicity. In these studies, we explore the use of the glutathione s-transferase (GST) to anchor the bactericidal peptide, melittin, to the surface of poly(ethylene glycol) diacrylate (PEGDA) hydrogel microspheres. We show that therapeutic levels of protein can be anchored to the surface of the microspheres using the GST anchor. We compared the therapeutic efficacy of recombinant melittin released from PEGDA microspheres to melittin. We found that, when released by an activating enzyme, thrombin, recombinant melittin efficiently inhibits growth of the pathogenic bacterium Streptococcus pyogenes as effectively as melittin created by solid phase peptide synthesis. We conclude that a GST protein anchor can be used to immobilize functional protein to PEGDA microspheres and the protein will remain immobilized under physiological conditions until the protein is enzymatically released