DOCSLIB.ORG

WO 2017/129998 Al 3 August 2017 (03.08.2017) P O P C T

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
WO 2017/129998 Al 3 August 2017 (03.08.2017) P O P C T (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2017/129998 Al 3 August 2017 (03.08.2017) P O P C T (51) International Patent Classification: (72) Inventors: CARPENTER, Byron; Medical Research C07K 14/47 (2006.01) Council, Polaris House, North Star Avenue, Swindon SN2 1FL (GB). LESLIE, Andrew; Medical Research Council, (21) International Application Number: Polaris House, North Star Avenue, Swindon SN2 1FL PCT/GB20 17/050221 (GB). NEHME, Rony; Medical Research Council, Polaris (22) International Filing Date: House, North Star Avenue, Swindon SN2 1FL (GB). 27 January 2017 (27.01 .2017) TATE, Christopher Gordon; Medical Research Council, Polaris House, North Star Avenue, Swindon SN2 1FL (25) Filing Language: English (GB). WARNE, Antony; Medical Research Council, Po (26) Publication Language: English laris House, North Star Avenue, Swindon SN2 1FL (GB). (30) Priority Data: (74) Agent: PEARS, Michael; Potter Clarkson LLP, The Bel- 1601 690.9 29 January 20 16 (29.01.2016) GB grave Centre, Talbot Street, Nottingham NG1 5GG (GB). (71) Applicant: HEPTARES THERAPEUTICS LIMITED (81) Designated States (unless otherwise indicated, for every [GB/GB]; BioPark, Broadwater Road, Welwyn Garden kind of national protection available): AE, AG, AL, AM, City Hertfordshire AL7 3AX (GB). AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. [Continued on nextpage] (54) Title: G PROTEINS (57) Abstract: The invention provides a mutant of a parent heterotrimeric G protein alpha (Ga) subunit, which mutant (i) lacks at least one helix of the helical domain of the parent Ga subunit; (ii) is capable of binding to a GPCR in the ab sence of a heterotrimeric G protein beta ( β) subunit and a heterotrimeric G protein gamma (Gy) subunit; and (iii) has an amino acid sequence that contains one or more mutations compared to the amino acid sequence of the parent heterotri meric Ga subunit, which mutations are selected from a dele tion, a substitution and an insertion. w o 2017/129998 Ai I il II II 11 I I 11 III III III lllll lllll lllll lllll lllll 111 llll 11llll (84) Designated States (unless otherwise indicated, for every Published: kind of regional protection available): ARIPO (BW, GH, — with international search report (Art. 21(3)) GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, — before the expiration of the time limit for amending the TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, claims and to be republished in the event of receipt of DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, ΓΤ , LT, amendments (Rule 48.2(h)) LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). G PROTEINS The present invention relates to mutant G proteins, and particularly to mutant alpha subunits of a heterotrimeric G protein. It also relates to products comprising such mutants, uses of the mutants, and methods involving such mutants. G proteins bind guanine nucleotides and act as molecular switches in a number of signalling pathways by interconverting between a GDP-bound inactive and a GTP-bound active state. They consist of two major classes: monomeric small G proteins and heterotrimeric G proteins. While small G proteins and the alpha subunit (Ga) of heterotrimeric G proteins both contain a GTPase domain (G-domain), Ga contains an additional helical domain (H-domain) and also forms a complex with G beta (G ) and G gamma (Gy) subunits. Although they undergo a similar signalling cycle, their activation differs in one important aspect. The guanine nucleotide exchange factors (GEFs) of small G proteins are largely cytosolic proteins, whereas the GEFs of Ga subunits are usually membrane-bound G protein coupled receptors (GPCRs). While GEFS of small G proteins interact directly with the GDP binding region, GPCRs bind to Ga at a site almost 30 A away from the GDP binding region and allosterically trigger GDP release to activate them. GPCRs constitute a very large family of proteins that control many physiological processes and are the targets of many effective drugs. Thus, they are of considerable pharmacological importance. Reference is made particularly to Overington et al (2006) Nature Rev. Drug Discovery 5, 993-996 which indicates that over a quarter of present drugs have a GPCR as a target. A list of GPCRs is given in Foord et al (2005) Pharmacol Rev. 57, 279-288, which is incorporated herein by reference. Three decades of biochemical and biophysical research have produced a model of G protein activation by GPCRs. Agonist binding to a GPCR induces subtle changes in the receptor structure 1 4, allowing a productive interaction to occur with the G protein. This process is likely to comprise at least two stages: an initial docking interaction, possibly involving the G protein βγ subunits or lipid moieties, induces a conformational change in the extreme C-terminus of the subunit5 6. The C-terminus, which is the major receptor-binding region7 and determinant of receptor specificity8,9 , is then able to fully engage the receptor. This interaction triggers mutually induced conformational changes in both the G protein and receptor 10 . In the G protein these changes are propagated to the nucleotide-binding pocket, resulting in the release of GDP10-1 1 . In the receptor the conformational changes feedback to the ligand-binding pocket, reducing the dissociation rate of the ligand, which results in significantly increased agonist binding affinity 2 13 . The mechanism of this affinity shift is likely to result from either subtle reorganisation of the ligand-binding pocket, or transition of the complex to a lower energy state due to the conformational stabilisation imparted by G protein binding. In this ternary complex the receptor acts as a chaperone, protecting the thermally labile nucleotide-free G protein from denaturation 14,15 . In the absence of guanine nucleotides this complex is stable; however in vivo, GTP is rapidly bound due to its high cellular concentration 12'14 . This triggers a conformational change, which causes dissociation of the G protein from the receptor 1 '17 , and separation of the Ga and βγ subunits 1416. The activated a-GTP and βγ subunits are then able stimulate their respective downstream signalling pathways. Atomic resolution mapping of the ligand-binding pocket is of significant importance for the design of drugs to modulate GPCR activity. Thus, methodology to crystallise receptors in their high-affinity agonist-bound conformation is a key prerequisite for efficient structure-based design of agonist compounds. To date, three approaches have accomplished this: first, the C-terminal peptide of transducin was crystallised in complex with both opsin 36 and metarhodopsin II37; second, a camelid antibody (Nb80), which induces the high-affinity agonist-bound state, has been crystallized in complex with 2A 38; third, heterotrimeric Gs has been crystallised in complex with 2A R . Despite the valuable insight into GPCR activation provided by these structures, they have several major disadvantages for wider structure-based drug design applications. The opsin and metarhodopsin II complexes were solved at 3.2 A and 2.85 A respectively, and in both cases electron density around the chromaphore-binding pocket was strong 36 7. However, the use of G protein C-terminal peptides to stabilise the active conformation of other GPCRs has been unsuccessful 10 . Furthermore, the conformational changes induced by the transducin peptide are much smaller than those observed in the P2AR-GS complex 10 , indicating that these structures represent an intermediate conformation along the activation pathway. The Nb80-fi2AR complex was solved at 3.5 A resolution, and also exhibited good electron density around the ligand-binding pocket 38 . However, the conformational changes induced by Nb80 are smaller than those observed in the 2AR- Gs complex 10 , suggesting that this structure may also represent an intermediate conformation. Furthermore, although Nb80 was derived specifically to bind AR and is therefore likely to efficiently couple to other closely related GPCRs (eg AR), new nanobodies likely need to be raised against more distantly related receptors. The P2AR- Gs complex was solved at 3.2 A resolution, however, in this structure electron density around the ligand-binding pocket was very poor. Furthermore, the complexity of crystallising G protein-GPCR complexes means this strategy is of limited use for wider structure-based drug design applications. All of the aforementioned complexes were solved at medium-high resolution, however, they provided insufficient detail around the ligand-binding pocket to accurately define the structural changes associated with the high-affinity agonist-bound conformation. Therefore, in order to accurately define these changes, and to allow optimal structure-based drug design, there is a strong requirement to solve the structures of G protein-GPCR complexes at greater than 2 A resolution. The separate GTPase and helical domains of the stimulatory G protein (Gas) have been previously transfected into COS-7 cells4 1 .
Load more

Recommended publications
  • Dynamin Functions and Ligands: Classical Mechanisms Behind
    1521-0111/91/2/123–134$25.00 http://dx.doi.org/10.1124/mol.116.105064 MOLECULAR PHARMACOLOGY Mol Pharmacol 91:123–134, February 2017 Copyright ª 2017 by The American Society for Pharmacology and Experimental Therapeutics MINIREVIEW Dynamin Functions and Ligands: Classical Mechanisms Behind Mahaveer Singh, Hemant R. Jadhav, and Tanya Bhatt Department of Pharmacy, Birla Institute of Technology and Sciences Pilani, Pilani Campus, Rajasthan, India Received May 5, 2016; accepted November 17, 2016 Downloaded from ABSTRACT Dynamin is a GTPase that plays a vital role in clathrin-dependent pathophysiology of various disorders, such as Alzheimer’s disease, endocytosis and other vesicular trafficking processes by acting Parkinson’s disease, Huntington’s disease, Charcot-Marie-Tooth as a pair of molecular scissors for newly formed vesicles originating disease, heart failure, schizophrenia, epilepsy, cancer, dominant ’ from the plasma membrane. Dynamins and related proteins are optic atrophy, osteoporosis, and Down s syndrome. This review is molpharm.aspetjournals.org important components for the cleavage of clathrin-coated vesicles, an attempt to illustrate the dynamin-related mechanisms involved phagosomes, and mitochondria. These proteins help in organelle in the above-mentioned disorders and to help medicinal chemists division, viral resistance, and mitochondrial fusion/fission. Dys- to design novel dynamin ligands, which could be useful in the function and mutations in dynamin have been implicated in the treatment of dynamin-related disorders. Introduction GTP hydrolysis–dependent conformational change of GTPase dynamin assists in membrane fission, leading to the generation Dynamins were originally discovered in the brain and identi- of endocytic vesicles (Praefcke and McMahon, 2004; Ferguson at ASPET Journals on September 23, 2021 fied as microtubule binding partners.
    [Show full text]
  • Highly Efficient Purification of Protein Complexes from Mammalian Cells
    Highly efficient purification of protein complexes from mammalian cells using a novel streptavidin-binding peptide and hexahistidine tandem tag system: Application to Bruton’s tyrosine kinase Yifeng Li,1 Sarah Franklin,1 Michael J. Zhang,1 and Thomas M. Vondriska1,2,3* 1Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, CA 2Department of Medicine/Cardiology, David Geffen School of Medicine, University of California, Los Angeles, CA 3Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, CA Received 2 June 2010; Revised 9 September 2010; Accepted 27 October 2010 DOI: 10.1002/pro.546 Published online 15 November 2010 proteinscience.org Abstract: Tandem affinity purification (TAP) is a generic approach for the purification of protein complexes. The key advantage of TAP is the engineering of dual affinity tags that, when attached to the protein of interest, allow purification of the target protein along with its binding partners through two consecutive purification steps. The tandem tag used in the original method consists of two IgG-binding units of protein A from Staphylococcus aureus (ProtA) and the calmodulin- binding peptide (CBP), and it allows for recovery of 20–30% of the bait protein in yeast. When applied to higher eukaryotes, however, this classical TAP tag suffers from low yields. To improve protein recovery in systems other than yeast, we describe herein the development of a three-tag system comprised of CBP, streptavidin-binding peptide (SBP) and hexa-histidine. We illustrate the application of this approach for the purification of human Bruton’s tyrosine kinase (Btk), which results in highly efficient binding and elution of bait protein in both purification steps (>50% recovery).
    [Show full text]
  • Crystal Structure of a Small G Protein in Complex with the Gtpase
    letters to nature of apolipoproteins2,12–14. However, our results show that the residues lipoprotein metabolism involving cell surface heparan sulfate proteoglycans. J. Biol. Chem. 269, 2764– 2+ 2772 (1994). in the conserved acidic motif of LR5 are buried to participate in Ca 16. Innerarity, T. L., Pitas, R. E. & Mahley, R. W. Binding of arginine-rich (E) apoprotein after coordination, instead of being exposed on the surface of the domain recombination with phospholipid vesicles to the low density lipoprotein receptors of fibroblasts. J. Biol. Chem. 254, 4186–4190 (1979). (Fig. 4a). Although lipoprotein uptake by members of the LDLR 17. Otwinowski, Z. & Minor, W. Data Collection and Processing (eds Sawyer, L., Isaacs, N. & Bailey, S.) family may involve an electrostatic component, perhaps through 556–562 (SERC Daresbury Laboratory, Warrington, UK, 1993). association of the lipoproteins with cell-surface proteoglycans15, the 18. CCP4. The SERC (UK) Collaborative Computing Project No. 4 A Suite of Programs for Protein Crystallography (SERC Daresbury Laboratory, Warrington, UK, 1979). LR5 structure demonstrates that the primary role for the conserved 19. Cowtan, K. D. Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography 31, 34–38 (1994). acidic residues in LDL-A modules is structural. It has been noted 20. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for binding protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 previously that only lipid-associated apolipoproteins bind with (1991). 16 high affinity to the LDLR ; the LR5 structure suggests an alternative 21.
    [Show full text]
  • Analysis of Proteins by Immunoprecipitation
    Laboratory Procedures, PJ Hansen Laboratory - University of Florida Analysis of Proteins by Immunoprecipitation P.J. Hansen1 1Dept. of Animal Sciences, University of Florida Introduction Immunoprecipitation is a procedure by which peptides or proteins that react specifically with an antibody are removed from solution and examined for quantity or physical characteristics (molecular weight, isoelectric point, etc.). As usually practiced, the name of the procedure is a misnomer since removal of the antigen from solution does not depend upon the formation of an insoluble antibody-antigen complex. Rather, antibody-antigen complexes are removed from solution by addition of an insoluble form of an antibody binding protein such as Protein A, Protein G or second antibody (Figure 1). Thus, unlike other techniques based on immunoprecipitation, it is not necessary to determine the optimal antibody dilution that favors spontaneously-occurring immunoprecipitates. Figure 1. Schematic representation of the principle of immunoprecipitation. An antibody added to a mixture of radiolabeled (*) and unlabeled proteins binds specifically to its antigen (A) (left tube). Antibody- antigen complex is absorbed from solution through the addition of an immobilized antibody binding protein such as Protein A-Sepharose beads (middle panel). Upon centrifugation, the antibody-antigen complex is brought down in the pellet (right panel). Subsequent liberation of the antigen can be achieved by boiling the sample in the presence of SDS. Typically, the antigen is made radioactive before the immunoprecipitation procedure, either by culturing cells with radioactive precursor or by labeling the molecule after synthesis has been completed (e.g., by radioiodination to iodinate tyrosine residues or by sodium [3H]borohydride reduction to label carbohydrate).
    [Show full text]
  • Ras Gtpase Chemi ELISA Kit Catalog No
    Ras GTPase Chemi ELISA Kit Catalog No. 52097 (Version B3) Active Motif North America 1914 Palomar Oaks Way, Suite 150 Carlsbad, California 92008, USA Toll free: 877 222 9543 Telephone: 760 431 1263 Fax: 760 431 1351 Active Motif Europe Waterloo Atrium Drève Richelle 167 – boîte 4 BE-1410 Waterloo, Belgium UK Free Phone: 0800 169 31 47 France Free Phone: 0800 90 99 79 Germany Free Phone: 0800 181 99 10 Telephone: +32 (0)2 653 0001 Fax: +32 (0)2 653 0050 Active Motif Japan Azuma Bldg, 7th Floor 2-21 Ageba-Cho, Shinjuku-Ku Tokyo, 162-0824, Japan Telephone: +81 3 5225 3638 Fax: +81 3 5261 8733 Active Motif China 787 Kangqiao Road Building 10, Suite 202, Pudong District Shanghai, 201315, China Telephone: (86)-21-20926090 Hotline: 400-018-8123 Copyright 2021 Active Motif, Inc. www.activemotif.com Information in this manual is subject to change without notice and does not constitute a commit- ment on the part of Active Motif, Inc. It is supplied on an “as is” basis without any warranty of any kind, either explicit or implied. Information may be changed or updated in this manual at any time. This documentation may not be copied, transferred, reproduced, disclosed, or duplicated, in whole or in part, without the prior written consent of Active Motif, Inc. This documentation is proprietary information and protected by the copyright laws of the United States and interna- tional treaties. The manufacturer of this documentation is Active Motif, Inc. © 2021 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150; Carlsbad, CA 92008.
    [Show full text]
  • Magresyn ® Protein G
    MagReSyn® Protein G Immobilized Protein G magnetic 1.4. Additional Equipment and Materials microparticles Magnetic separator, Vortex mixer, Buffers and solutions, end-over-end mixer (optional) Ordering Information Cat. No. Quantity 2. Immunoglobulin Purification Factors that may affect the attachment of antibodies include the isotype of the MR-PRG002 2 ml immunoglobulin, buffer composition and pH, and the presence of MR-PRG005 5 ml contaminants/interfering compounds. The quantity of microparticles needs to be optimized for each individual application. We recommend the application of excess MR-PRG010 2 x 5 ml ligand to ensure saturation of the Protein G microparticles. The binding efficiency can be determined by comparing the ligand concentration before and after coupling. This product is for research use only MagReSyn® Protein G is compatible with various commonly used buffers, including Tris and Phosphate. Recommended buffers include: Binding/wash buffer - TBS (50 mM Tris pH 7.5, 150 mM NaCl, 0.025% Tween® 20) or PBS (50 mM Phosphate pH 7.5, 150 mM Table of Contents: NaCl, 0.025% Tween® 20); Elution Buffer (Native): 0.1 M glycine pH 2.5 or 2.5% acetic 1. Product Description acid; Elution Buffer (Denaturing): SDS-PAGE electrophoresis buffer. 2. Immunoglobulin Purification 3. Immunoprecipitation NOTE: All reagents should be freshly prepared and of analytical grade to ensure 4. Recommended Storage optimal performance. The procedures, methods and buffer solutions described below serve as an example and are not intended to be limiting. MagReSyn® Protein G is 5. Antibody Binding Guide compatible with a range of different buffers for binding of antibodies.
    [Show full text]
  • RGS22 Antibody (Pab)
    21.10.2014RGS22 antibody (pAb) Rabbit Anti-Human/Mouse/Rat Regulator of G-protein signaling 22 (PRTD -NY2) Instruction Manual Catalog Number PK-AB718-7001 Synonyms RGS22 Antibody: Regulator of G-protein signaling 22, PRTD-NY2 Description Regulator of G-protein signaling (RGS) proteins contain an 120 amino acid conserved domain, termed the RGS domain, that acts as a GTPase-activating protein that acts to reduce the signal transmitted by the receptor-activated G-alpha subunit. RGS22 is a recently identified member of this family that localizes to the testis and can interact with guanine nucleotide binding proteins alpha 11, 12, and 13 (GNA11, GNA12, and GNA13). While RGS22 has been postulated to play a role in spermiogenesis in the testis, it is also expressed in several cancer cell lines with an epithelial origin and associated with cancer metastasis. Its overexpression in a highly metastatic cancer causes a decrease in cell migration and a reduction of the invasive potential of the cells, suggesting that RGS22 may be a potential prognostic biomarker for metastasis. Quantity 100 µg Source / Host Rabbit Immunogen RGS22 antibody was raised against a 19 amino acid synthetic peptide near the amino terminus of human RGS22. Purification Method Affinity chromatography purified via peptide column. Clone / IgG Subtype Polyclonal antibody Species Reactivity Human, Mouse, Rat Specificity At least four isoforms of RGS22 are known to exist; this antibody will detect the three longest isoforms. RGS22 antibody is predicted to not cross-react with other RGS proteins Formulation Antibody is supplied in PBS containing 0.02% sodium azide. Reconstitution During shipment, small volumes of antibody will occasionally become entrapped in the seal of the product vial.
    [Show full text]
  • Plasma Membrane Repairs by Small Gtpase Rab3a
    Published June 20, 2016 JCB: SpotlightReview Plasma membrane repairs by small GTPase Rab3a Camilla Raiborg1,2 and Harald Stenmark1,2 1Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, Montebello, N-0379 Oslo, Norway 2Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Montebello, N-0379 Oslo, Norway Lysosomes fuse with the plasma membrane to help repair in lysosome-mediated PM repair has not been addressed. In this membrane lesions, but how they are positioned close to issue, Encarnação et al. screened siRNAs that target human Rab GTPases to identify Rabs involved in translocating lysosomes to these lesions is not fully understood. Now, Encarnação et the PM in response to the Ca2+ ionophore ionomycin and in the al. (2016. J. Cell Biol. http ://dx .doi .org /10 .1083 /jcb repair of PM holes caused by the bacterial toxin Streptolysin-O .201511093) demonstrate that the lysosomal GTPase (Encarnação et al., 2016). One of the most prominent hits in this Rab3a and its effectors orchestrate lysosome positioning screen was Rab3a, a GTPase previously characterized mainly in the regulation of Ca2+-induced exocytosis of synaptic vesicles and plasma membrane repair. in neurons and of dense-core vesicles in endocrine cells (Gep- pert et al., 1994; Johannes et al., 1994). Even though Rab3a is Downloaded from When the plasma membrane (PM) of eukaryotic cells is predominantly expressed in neurons and endocrine tissues, En- wounded by mechanical damage or by bacterial toxins, the carnação et al. (2016) were able to detect its expression in HeLa wound is normally repaired within seconds by mechanisms cells and melanocytes.
    [Show full text]
  • G Protein Regulation of MAPK Networks
    Oncogene (2007) 26, 3122–3142 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc REVIEW G Protein regulation of MAPK networks ZG Goldsmith and DN Dhanasekaran Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA, USA G proteins provide signal-coupling mechanisms to hepta- the a-subunits has been used as a basis for the helical cell surface receptors and are criticallyinvolved classification of G proteins into Gs,Gi,Gq and G12 in the regulation of different mitogen-activated protein families in which the a-subunits that show more than kinase (MAPK) networks. The four classes of G proteins, 50% homology are grouped together (Simon et al., defined bythe G s,Gi,Gq and G12 families, regulate 1991). In G-protein-coupled receptor (GPCR)-mediated ERK1/2, JNK, p38MAPK, ERK5 and ERK6 modules by signaling pathways, ligand-activated receptors catalyse different mechanisms. The a- as well as bc-subunits are the exchange of the bound GDP to GTP in the a-subunit involved in the regulation of these MAPK modules in a following which the GTP-bound a-subunit disassociate context-specific manner. While the a- and bc-subunits from the receptor as well as the bg-subunit. The GTP- primarilyregulate the MAPK pathwaysvia their respec- bound a-subunit and the bg-subunit stimulate distinct tive effector-mediated signaling pathways, recent studies downstream effectors including enzymes, ion channels have unraveled several novel signaling intermediates and small GTPase, thus regulating multiple signaling including receptor tyrosine kinases and small GTPases pathways including those involved in the activation of through which these G-protein subunits positivelyas well mitogen-activated protein kinase (MAPK) modules as negativelyregulate specific MAPK modules.
    [Show full text]
  • 6511-Protein G-Sepharose
    FOR RESEARCH USE ONLY! Protein G-Sepharose rev. 09/16 ° Store at 4 C. Do not freeze. Cat. No. 6511-1 Protein G-Sepharose, 1 ml settled resin 6511-5 Protein G-Sepharose, 5 ml settled resin 6511-25 Protein G-Sepharose, 25 ml settled resin 6511-100 Protein G-Sepharose, 100 ml settled resin 6511-1000 Protein G-Sepharose, 1 L settled resin Support: 6% cross-linked Sepharose beads supplied as 50% slurry (e.g., 1 ml of settled resin is equivalent to 2 ml of 50% slurry) in 20% Ethanol/H2O. Binding Capacity: >20 mg human or rabbit IgG/ml of settled resin. Flow Rate Tested*: 0.85 cm/min. *Test condition: Linear flow rate determined in 2 ml column with internal diameter of 1.5 cm. Introduction: Protein G is a cell wall protein produced by group G streptococcus. Like protein A, this bacteria-derived protein binds with high affinity & specificity to the Fc portion of most mammalian immunoglobulins. Therefore, Protein G has been widely used for IgG purification. BioVision’s Protein G (Cat. # 6510) is a genetically engineered protein containing three Ig-binding regions of native Protein G. The cell wall binding region, albumin binding region and other non-specific regions have been eliminated from the recombinant Protein G to ensure the maximum specific IgG binding. The coupling technique is optimized to give a higher binding capacity for IgG & minimum leaching of recombinant Protein G. In addition, Protein G-Sepharose beads display high chemical & physical stability as well as high flow rate, hydrophilicity & high gel strength.
    [Show full text]
  • (12) United States Patent (10) Patent No.: US 9.410,182 B2 W (45) Date of Patent: Aug
    USOO941 0182B2 (12) United States Patent (10) Patent No.: US 9.410,182 B2 W (45) Date of Patent: Aug. 9, 2016 (54) PHOSPHATASE COUPLED OTHER PUBLICATIONS GLYCOSYLTRANSFERASE ASSAY Wu et al., R&D Systems Poster, “Universal Phosphatase-Coupled (75) Inventor: Zhengliang L. Wu, Edina, MN (US) Glycosyltransferase Assay”, Apr. 2010, 3 pages.* “Malachite Green Phosphate Detection Kit'. Research and Diagnos (73) Assignee: Bio-Techne Corporation, Minneapolis, tic Systems, Inc. Catalog No. DY996, Feb. 4, 2010, 6 pages.* MN (US) Zhu et al., Analytica Chimica Acta 636:105-110, 2009.* Motomizu et al., Analytica Chimica Acta 211:119-127, 1988.* *) NotOt1Ce: Subjubject to anyy d1Sclaimer,disclai theh term off thisthi Schachter et al., Methods Enzymol. 98.98-134, 1983.* patent is extended or adjusted under 35 Unverzagt et al., J. Am. Chem. Soc. 112:9308-9309, 1990.* U.S.C. 154(b) by 0 days. IUBMB Enzyme Nomenclature for EC 3.1.3.1, obtained from www. chem.cmul.ac.uk/iubmb? enzyme/EC3/1/3/5.html, last viewed on (21) Appl. No.: 13/699,175 Apr. 13, 2015, 1 page.* IUBMB Enzyme Nomenclature for EC 3.1.3.5, obtained from www. (22) PCT Filed: May 24, 2010 chem.cmul.ac.uk/iubmb? enzyme/EC3/1/3/5.html, last viewed on Apr. 13, 2015, 1 page.* (86). PCT No.: PCT/US2010/035938 Compain et al., Bio. Med. Chem. 9:3077-3092, 2001.* Lee et al., J. Biol. Chem. 277:49341-49351, 2002.* S371 (c)(1), Donovan R S et al., “A Solid-phase glycosyltransferase assay for (2), (4) Date: Nov.
    [Show full text]
  • Subcellular Localization and Quantitation of the Major Neutrophil Pertussis Toxin Substrate, G N Gary M
    Subcellular Localization and Quantitation of the Major Neutrophil Pertussis Toxin Substrate, G n Gary M. Bokoch, Kevin Bickford, and Benjamin P. Bohl Department of Immunology, Research Institute of Scripps Clinic, La Jolla, California 92037 Abstract. The subcellular distribution of G protein ules contained detectable G protein. Based on the abil- subunits in the neutrophil was examined. Cells were ity of exogenous G protein beta/gamma subunits to in- nitrogen cavitated and subcellular organelles fraction- crease the ADP ribosylation of the cytosolic form of G ated on discontinuous sucrose gradients. The presence protein and upon the hydrodynamic behavior of the of GTP-binding regulatory protein (G protein) alpha cytosolic protein, it is likely that this represents an un- and beta/gamma subunits in each organelle was de- complexed G protein alpha subunit. Proteolytic map- termined using three methods of analysis: specific ping with Staphylococcus aureus V8 protease suggests binding of guanine nucleotide, ADP ribosylation by the soluble alpha subunit is from G,, the major per- pertussis toxin, and immunoblot analysis with subunit- tussis toxin substrate of human neutrophils. Using specific G protein antibodies. Both plasma membrane quantitative analysis, the levels of the 40-kD G protein and cytosolic G protein components were detected. In alpha subunit and of the 35/36-kD beta subunit in the contrast, neither the specific nor the azurophilic gran- neutrophil membrane were determined. r~E GTP-binding regulatory proteins (G proteins) j be involved in coupling the alpha subunits to membrane consist of a family of highly homologous proteins receptors (9) and, potentially, the membrane itself.
    [Show full text]