Fc-GDF15 glyco-engineering and receptor binding affinity optimization for body weight regulation.

GDF15 is a distant TGF-β family member that induces anorexia and weight loss. Due to its function, GDF15 has attracted attention as a potential therapeutic for the treatment of obesity and its associated metabolic diseases. However, the pharmacokinetic and physicochemical properties of GDF15 present several challenges for its development as a therapeutic, including a short half-life, high aggregation propensity, and protease susceptibility in serum.

Development of PF-06671008, a Highly Potent Anti-P-cadherin/Anti-CD3 Bispecific DART Molecule with Extended Half-Life for the Treatment of Cancer.

Bispecific antibodies offer a promising approach for the treatment of cancer but can be challenging to engineer and manufacture. Here we report the development of PF-06671008, an extended-half-life dual-affinity re-targeting (DART) bispecific molecule against P-cadherin and CD3 that demonstrates antibody-like properties. Using phage display, we identified anti-P-cadherin single chain Fv (scFv) that were subsequently affinity-optimized to picomolar affinity using stringent phage selection strategies, resulting in low picomolar potency in cytotoxic T lymphocyte (CTL) killing assays in the DART format.

A Combination of Structural and Empirical Analyses Delineates the Key Contacts Mediating Stability and Affinity Increases in an Optimized Biotherapeutic Single-chain Fv (scFv).

Fully-human single-chain Fv (scFv) proteins are key potential building blocks of bispecific therapeutic antibodies, but they often suffer from manufacturability and clinical development limitations such as instability and aggregation. The causes of these scFv instability problems, in proteins that should be theoretically stable, remains poorly understood. To inform the future development of such molecules, we carried out a comprehensive structural analysis of the highly stabilized anti-CXCL13 scFv E10.

Interleukin 1/Toll-like receptor-induced autophosphorylation activates interleukin 1 receptor-associated kinase 4 and controls cytokine induction in a cell type-specific manner.

IRAK4 is a central kinase in innate immunity, but the role of its kinase activity is controversial. The mechanism of activation for IRAK4 is currently unknown, and little is known about the role of IRAK4 kinase in cytokine production, particularly in different human cell types. We show IRAK4 autophosphorylation occurs by an intermolecular reaction and that autophosphorylation is required for full catalytic activity of the kinase.

Novel synthesis and structural characterization of a high-affinity paramagnetic kinase probe for the identification of non-ATP site binders by nuclear magnetic resonance.

To aid in the pursuit of selective kinase inhibitors, we have developed a unique ATP site binder tool for the detection of binders outside the ATP site by nuclear magnetic resonance (NMR). We report here the novel synthesis that led to this paramagnetic spin-labeled pyrazolopyrimidine probe (1), which exhibits nanomolar inhibitory activity against multiple kinases. We demonstrate the application of this probe by performing NMR binding experiments with Lck and Src kinases and utilize it to detect the binding of two compounds proximal to the ATP site.

H6PDH interacts directly with 11beta-HSD1: implications for determining the directionality of glucocorticoid catalysis.

Tissue specific amplification of glucocorticoid action through NADPH-dependent reduction of inactive glucocorticoid precursors by 11beta-hydroxysteroid dehydrogenase (11beta-HSD1) contributes to the development of visceral obesity, insulin resistance and Type 2 Diabetes. Hexose-6-phosphate dehydrogenase (H6PDH) is believed to supply NADPH for the reductase activity of 11beta-HSD1 in the lumen of the endoplasmic reticulum (ER), where the two enzymes are co-localized. We report here expression and purification of full-length and truncated N-terminal domain (NTD) of H6PDH in a mammalian expression system.

Investigation of leucine-rich repeat kinase 2 : enzymological properties and novel assays.

Mutations in leucine-rich repeat kinase 2 (LRRK2) comprise the leading cause of autosomal dominant Parkinson's disease, with age of onset and symptoms identical to those of idiopathic forms of the disorder. Several of these pathogenic mutations are thought to affect its kinase activity, so understanding the roles of LRRK2, and modulation of its kinase activity,may lead to novel therapeutic strategies for treating Parkinson's disease. In this study, highly purified, baculovirus-expressed proteins have been used,for the first time providing large amounts of protein that enable a thorough enzymatic characterization of the kinase activity of LRRK2.

Mass spectrometry-compatible silver staining.

INTRODUCTIONThe ammoniacal silver staining method is one of the most sensitive methods used to detect proteins on an SDS-PAGE gel. However, this and other standard silver staining methods are not compatible with mass spectrometry (MS), which is fast becoming the best way to identify proteins isolated on 2D gels. Because the proteins in gels to be analyzed by mass spectroscopy cannot be modified, many of the common sensitizing agents (e.

Preparative 2D Gel Electrophoresis with Immobilized pH Gradients: IPG Strip Equilibration.

INTRODUCTIONThe equilibration step serves to saturate the IPG strip with the SDS buffer system required for the second-dimension separation. The equilibration solution consists of buffer, urea, glycerol, reductant, SDS, and dye. The buffer (50 mM Tris-HCl, pH 8.

Preparation of Vertical SDS Slab Gels: Simultaneous Casting of Multiple-Gradient Gels.

INTRODUCTIONGradient SDS-PAGE gels provide the best resolution over a wide range of molecular weights, resulting in sharper protein spots, because diffusion is minimized by the decreasing pore size in the gel. However, gradient gels are more difficult to produce reproducibly; thus, they are commonly cast with multiple-gel casters, which allows for an identical set of gels to be produced for an experiment. Presented here is a method for casting gradient gels using a multiple-gel casting system.

Preparation of Vertical SDS Slab Gels: Casting a Single Homogeneous Gel.

INTRODUCTIONFollowing the separation of proteins by IEF, the second dimension is carried out by SDS-PAGE. This protocol details the method for casting single homogeneous SDS-PAGE gels. Homogeneous gels (with the same %T and %C throughout) offer the best resolution for a particular molecular-weight range and are commonly used because they are the easiest to pour reproducibly.

Phosphoprotein Staining with the GelCode Phosphoprotein Staining Kit.

INTRODUCTIONThe phosphorylation state of a protein has an important role in the regulation of a wide variety of cellular processes. As a result, there has been a great deal of interest in detecting phosphorylated proteins. The method presented here uses the GelCode phosphoprotein staining kit (Pierce Chemical Company).

Preparative 2D Gel Electrophoresis with Immobilized pH Gradients: IEF of Proteins in an IEF-Dedicated Electrophoresis Unit.

INTRODUCTIONThis protocol describes a method for separating proteins based on their net charge using the technique of isoelectric focusing (IEF) on immobilized pH gradient (IPG) gels, providing the first dimension of the 2D separation. In this protocol, the IPG gels are focused using self-contained instruments for IEF. These high-voltage systems allow fewer manipulations of the IPG gels, resulting in less error, strip mix-up, contamination, air contact, or urea crystallization.

Preparative 2D Gel Electrophoresis with Immobilized pH Gradients: Isoelectric Focusing of Proteins in a Multipurpose Flatbed Electrophoresis Unit.

INTRODUCTIONThis protocol describes a method for separating proteins based on their net charge using the technique of isoelectric focusing (IEF) on immobilized pH gradient (IPG) gels. This method serves as the first dimension of the 2D separation. The method described in this protocol utilizes a flatbed unit; however, self-contained instruments for IEF are also available.

Preparative 2D Gel Electrophoresis with Immobilized pH Gradients: Rehydration of IPG Strips for Isoelectric Focusing of Proteins.

INTRODUCTIONThis protocol describes a method for rehydration of IPG gel strips in preparation for their use for isoelectric focusing (IEF) on immobilized pH gradient (IPG) gels. Following rehydration, IEF can be performed using either a flatbed unit or a self-contained instrument.

Preparative 2D Gel Electrophoresis with Immobilized pH Gradients: SDS-PAGE of Proteins.

INTRODUCTIONFollowing first-dimension IEF and equilibration of the IPG gel strips, the proteins are separated on the basis of their molecular weight in the second dimension on an SDS-PAGE gel. Systems for this separation are available from a variety of suppliers and are commonly found in many protein chemistry laboratories. This protocol describes a method for placement of the IPG strip and gives some recommended electrophoresis conditions for these second-dimension gels.

Staining membrane-bound proteins with colloidal gold.

INTRODUCTIONColloidal Gold is the most sensitive staining technique for proteins bound on membranes, detecting as little as 1-3 ng of protein. Protein spots are permanently stained a dark red after incubation with the Colloidal Gold solution. Colloidal Gold staining can detect proteins on both nitrocellulose and PVDF membranes, but it is not recommended for nylon membranes.

Staining membrane-bound proteins with coomassie blue r250.

INTRODUCTIONCoomassie Blue R250 permanently stains membrane-bound proteins and is compatible with PVDF and nitrocellulose membranes, but it is incompatible with nylon membranes. This technique is relatively insensitive, with a detection limit of ~1.5 μg of protein.

Staining membrane-bound proteins with ponceau s.

INTRODUCTIONBecause Ponceau S is relatively insensitive (~1 μg of protein), only the most abundant proteins will be visible. However, it is a reversible stain that can be removed completely with H(2)O prior to processing the blots. After staining, a soft lead pencil can be used to record the presence of visible proteins and molecular-weight markers, which will help when aligning the proteins detected on the membrane by western analysis with those in a total protein-stained gel or membrane.

Methods for the differential integrative omic analysis of plasma from a transgenic disease animal model.

Multitiered quantitative analysis of biological systems is rapidly becoming the desired approach to study hierarchical functional interactions between proteins and metabolites. We describe here a novel systematic approach to analyze organisms with complex metabolic regulatory networks. By using precise analytical methods to measure biochemical constituents and their relative abundance in whole plasma of transgenic ApoE*3-Leiden mice and an isogenic wild-type control group, simultaneous snapshots of metabolic and protein states were obtained.

Integrative biological analysis of the APOE*3-leiden transgenic mouse.

Integrative (or systems biology) is a new approach to analyzing biological entities as integrated systems of genetic, genomic, protein, metabolite, cellular, and pathway events that are in flux and interdependent. Here, we demonstrate the application of intregrative biological analysis to a mammalian disease model, the apolipoprotein E3-Leiden (APO*E3) transgenic mouse. Mice selected for the study were fed a normal chow diet and sacrificed at 9 weeks of age-conditions under which they develop only mild type I and II atherosclerotic lesions.