DOCSLIB.ORG

(12) Patent Application Publication (10) Pub. No.: US 2011/0045572 A1 Roggen Et Al

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
(12) Patent Application Publication (10) Pub. No.: US 2011/0045572 A1 Roggen Et Al US 2011 0045572A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0045572 A1 Roggen et al. (43) Pub. Date: Feb. 24, 2011 (54) PROTEIN VARANTS HAVING MODIFIED (30) Foreign Application Priority Data IMMUNOGENICITY Apr. 28, 2000 (DK). ... PA 2000 OO707 (75) Inventors: Erwin Ludo Roggen, Lyngby Feb. 28, 2001 (DK) ........................... PA 2001 OO327 (DK); Steffen Ernst, Bronshoi Publication Classification (DK); Allan Svendsen, Horsholm (51) Int. Cl. (DK); Esben Peter Friis, Valby CI2N 9/56 (2006.01) (DK); Claus Von Der Osten, CI2N 9/48 (2006.01) Lyngby (DK) CI2N 15/63 (2006.01) CI2N 5/10 (2006.01) Correspondence Address: CI2N L/15 (2006.01) NOVOZYMES NORTHAMERICA, INC. (52) U.S. Cl. ...................... 435/222:435/212:435/320.1; 500 FIFTHAVENUE, SUITE 1600 435/325; 435/348; 435/419; 435/254.11 NEW YORK, NY 10110 (US) (57) ABSTRACT (73) Assignee: NOVOZYMESA/S, BAGSVAERD The present invention relates to a method of selecting a pro (DK) tein variant having modified immunogenicity as compared to the parent protein comprising the steps obtaining antibody (21) Appl. No.: 12/699,979 binding peptide sequences, using the sequences to localise epitope sequences on the 3-dimensional structure of parent Filed: Feb. 4, 2010 protein, defining an epitope area including amino acids situ (22) ated within 5 A from the epitope amino acids constituting the epitope sequence, changing one or more of the amino acids Related U.S. Application Data defining the epitope area of the parent protein by genetical (63) Continuation of application No. 09/957,806, filed on engineering mutations of a DNA sequence encoding the par Sep. 21, 2001, now abandoned, Continuation of appli ent protein, introducing the mutated DNA sequence into a cation No. PCT/DK01/00293, filed on Apr. 30, 2001. Suitable host, culturing said host and expressing the protein variant, and evaluating the immunogenicity of the protein (60) Provisional application No. 60/203,345, filed on May variant using the parent protein as reference. The invention 10, 2000, provisional application No. 60/277,817, further relates to the protein variant and use thereof, as well as filed on Mar. 21, 2001. to a method for producing said protein variant. men BPN' on Alcatase amoa BAP-R oxer Savinase mom Esperase rCrPD498 49 99 149 199 249 Patent Application Publication Feb. 24, 2011 US 2011/0045572 A1 2O 18 16 14 geese. BPN' 12 as Acalase O alo BAP-R oxane Savinase 8 -O-Esperase 6 a-CoPD498 4. 2 as S . OOOOO O Rice (CCC(Oc CCC c cCcCCO 1 49 99 149 199 249 FIG. 1 US 2011/0045572 A1 Feb. 24, 2011 PROTEIN VARLANTS HAVING MODIFIED Such an epitope, maybe converting it from a high affinity to a IMMUNOGENICITY low affinity epitope, or maybe even result in epitope loss, i.e. that the epitope cannot sufficiently bind an antibody to elicit CROSS-REFERENCE TO RELATED an immunogenic response. APPLICATIONS 0007. There is a need for methods to identify epitopes on 0001. This application is a continuation of U.S. applica proteins and alter these epitopes in order to modify the immu tion Ser. No. 09/957,806 filed on Sep. 21, 2001, which is a nogenicity of proteins in a targeted manner. Such methods continuation of PCT/DK01/00293 filed Apr. 30, 2001 and and kits for their execution can have at least four useful claims, under 35 U.S.C. 119, priority or the benefit of Danish purposes: application nos. PA 2000 00707 and PA 2001 00327 filed Apr. 0008 1) reduce the allergenicity of a commercial protein 28, 2000 and Feb. 28, 2001, respectively, and U.S. application using protein engineering. Nos. 60/203,345 and 60/277,817 filed May 10, 2000 and Mar. 0009. 2) reduce the potential of commercial proteins to 21, 2001, respectively, the contents of which are fully incor cross-react with environmental allergens and hence cause porated herein by reference. allergic reactions in people sensitized to the environmental allergens (or vice versa). FIELD OF INVENTION 0010 3) improve the immunotherapeutic effect of allergen vaccines. 0002 The present invention relates to a method of select 00.11 4) assist characterization of clinical allergies in ing a protein variant having modified immunogenicity as order to select the appropriate treatment, including allergen compared to the parent protein, to the protein variant and use vaccination. thereof, as well as to a method for producing said protein (0012. In WO99/53038 (Genencor Int.) as well as in prior variant. references (Kammereretal, Clin. Exp. Allergy, 1997, Vol.27, pp. 1016-1026: Sakakibara et al., J. Vet. Med. Sci., 1998; Vol. BACKGROUND OF THE INVENTION 60, pp. 599–605), methods are described, which identify lin 0003. An increasing number of proteins, including ear T-cell epitopes among a library of known peptide enzymes, are being produced industrially, for use in various sequences, each representing part of the primary sequence of industries, housekeeping and medicine. Being proteins they the protein of interest. Further, several similar techniques for are likely to stimulate an immunological response in man and localization of B-cell epitopes are disclosed by Walshet et al. animals, including an allergic response. J. Immunol. Methods, vol. 121, 1275-280, (1989), and by 0004 Depending on the application, individuals get sen Schoofs et al. J. Immunol. vol. 140, 611-616, (1987). All of sitised to the respective allergens by inhalation, direct contact these methods, however, only leads to identification of linear with skin and eyes, or injection. The general mechanism epitopes, not to identification of structural or discontinu behind an allergic response is divided in a sensitisation phase ous epitopes, which are found on the 3-dimensional Surface and a symptomatic phase. The sensitisation phase involves a of protein molecules and which comprise amino acids from first exposure of an individual to an allergen. This event several discrete sites of the primary sequence of the protein. activates specific T- and B-lymphocytes, and leads to the For several allergens, it has been realized that the dominant production of allergen specific IgE antibodies (in the present epitopes are of such discontinuous nature (Collins et al., Clin. context the antibodies are denoted as usual, i.e. immunoglo Exp. All. 1996, vol. 26, pp. 36-42). bulin E IgE etc.). These IgE antibodies eventually facilitate 0013 Slootstra et al; Molecular Diversity, 2, pp. 156-164, allergen capturing and presentation to T-lymphocytes at the 1996 disclose the screening of a semi-random library of syn onset of the symptomatic phase. This phase is initiated by a thetic peptides for their binding properties to three mono second exposure to the same or a resembling antigen. The clonal antibodies by immobilizing the peptides on polyethyl specific IgE antibodies bind to the specific IgE receptors on ene pins and binding a dilution series of each antibody to the mast cells and basophils, among others, and capture at the pins. This reference does not disclose any indication of how same time the allergen. The polyclonal nature of this process the antibody binding peptide sequences relate to any full results in bridging and clustering of the IgE receptors, and protein antigens or allergens. Subsequently in the activation of mast cells and basophils. (0014. In WO92/10755 a method for modifying proteins to This activation triggers the release of various chemical obtain less immunogenic variants is described. Randomly mediators involved in the early as well as late phase reactions constructed protein variants, revealing a reduced binding of of the symptomatic phase of allergy. Prevention of allergy in antibodies to the parent enzyme as compared to the parent Susceptible individuals is therefore a research area of great enzyme itself, are selected for the measurement in animal importance. models in terms of allergenicity. Finally, it is assessed 0005 For certain forms of IgE-mediated allergies, a whether reduction in immunogenicity is due to true elimina therapy exists, which comprises repeated administration of tion of an epitope or a reduction in affinity for antibodies. This allergen preparations called allergen vaccines (Int. Arch. method targets the identification of amino acids that may be Allergy Immunol., 1999, Vol. 119, pp 1-5). This leads to part of structural epitopes by using a complete protein for reduction of the allergic symptoms, possibly due to a redirec assessing antigen binding. The major drawbacks of this tion of the immune response away from the allergic (Th2) approach are the trial and error character, which makes it a pathway and towards the immunoprotective (Th1) pathway lengthy and expensive process, and the lack of general infor (Int. Arch. Allergy Immunol., 1999, Vol. 119, pp 1-5). mation on the epitope patterns. Without this information, the 0006 Various attempts to reduce the immunogenicity of results obtained for one protein can not be applied on another polypeptides and proteins have been conducted. It has been protein. found that Small changes in an epitope may affect the binding 10015 WO99/47680 (ALK-ABELLO) discloses the iden to an antibody. This may result in a reduced importance of tification and modification of B-cell epitopes by protein engi US 2011/0045572 A1 Feb. 24, 2011 neering. However, the method is based on crystal structures of 0030. In accordance with the present invention there may Fab-antigen complexes, and B-cell epitopes are defined as “a be employed conventional molecular biology, microbiology, section of the Surface of the antigen comprising 15-25 amino and recombinant DNA techniques within the skill of the art. acid residues, which are within a distance from the atoms of Such techniques are explained fully in the literature. See, e.g., the antibody enabling direct interaction” (p.
Load more

Recommended publications
  • International Journal of Advanced and Applied Sciences, 4(12) 2017, Pages: 31-35
    International Journal of Advanced and Applied Sciences, 4(12) 2017, Pages: 31-35 Contents lists available at Science-Gate International Journal of Advanced and Applied Sciences Journal homepage: http://www.science-gate.com/IJAAS.html Tertiary structure prediction of bromelain from Ananas Comosus using comparative modelling method Fatahiya Mohamed Tap 1, Fadzilah Adibah Abd Majid 2, *, Nurul Bahiyah Ahmad Khairudin 1 1Chemical Energy Conversion and Applications Group, Malaysia Japan International Institute of Technology, Universiti Teknologi Malaysia, Kuala Lumpur, Malaysia 2Institute of Marine Biotechnology, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Malaysia ARTICLE INFO ABSTRACT Article history: Bromelain is a general name for a family of sulfhydryl which can be found in Received 21 February 2017 the proteolytic enzyme group from pineapples (Ananas Comosus). This study Received in revised form focuses on the prediction of three dimensional structures (3D) of stem 2 October 2017 bromelain using the method of comparative modelling. The amino acid Accepted 8 October 2017 sequence of bromelain was obtained from the NCBI database was used as a tool to search for proteins with known 3D structures related to the target Keywords: sequence. Suitable template was chosen based on >30% sequence similarity Bromelain and lowest e-value. Based on these criteria, 1YAL was selected as the best Comparative modelling template with 55% of sequence similarity and 9x10-52 of e-value. Sequence alignment © 2017 The Authors. Published by IASE. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction industrial and pharmacology area, therefore it is essential to maintain and improve bromelain *Bromelain can be derived from stem and fruit of properties in conformation and catalytic activity in pineapples.
    [Show full text]
  • Concentrate of Proteolytic Enzymes Enriched in Bromelain
    20 September 2012 EMA/648483/2012 Committee for Medicinal Products for Human Use (CHMP) Assessment report NexoBrid Concentrate of proteolytic enzymes enriched in bromelain Procedure No. EMEA/H/C/002246 Note Assessment report as adopted by the CHMP with all information of a commercially confidential nature deleted. 7 Westferry Circus ● Canary Wharf ● London E14 4HB ● United Kingdom Telephone +44 (0)20 7418 8400 Facsimile +44 (0)20 7418 8416 E-mail [email protected] Website www.ema.europa.eu An agency of the European Union © European Medicines Agency, 2012. Reproduction is authorised provided the source is acknowledged. Table of contents 1. Background information on the procedure .............................................. 5 1.1. Submission of the dossier.................................................................................... 5 1.2. Steps taken for the assessment of the product ....................................................... 6 2. Scientific discussion ................................................................................ 7 2.1. Introduction ...................................................................................................... 7 2.2. Quality aspects .................................................................................................. 9 2.3. Non-clinical aspects .......................................................................................... 20 2.4. Clinical aspects ................................................................................................ 29 2.5.
    [Show full text]
  • Chapter 11 Cysteine Proteases
    CHAPTER 11 CYSTEINE PROTEASES ZBIGNIEW GRZONKA, FRANCISZEK KASPRZYKOWSKI AND WIESŁAW WICZK∗ Faculty of Chemistry, University of Gdansk,´ Poland ∗[email protected] 1. INTRODUCTION Cysteine proteases (CPs) are present in all living organisms. More than twenty families of cysteine proteases have been described (Barrett, 1994) many of which (e.g. papain, bromelain, ficain , animal cathepsins) are of industrial impor- tance. Recently, cysteine proteases, in particular lysosomal cathepsins, have attracted the interest of the pharmaceutical industry (Leung-Toung et al., 2002). Cathepsins are promising drug targets for many diseases such as osteoporosis, rheumatoid arthritis, arteriosclerosis, cancer, and inflammatory and autoimmune diseases. Caspases, another group of CPs, are important elements of the apoptotic machinery that regulates programmed cell death (Denault and Salvesen, 2002). Comprehensive information on CPs can be found in many excellent books and reviews (Barrett et al., 1998; Bordusa, 2002; Drauz and Waldmann, 2002; Lecaille et al., 2002; McGrath, 1999; Otto and Schirmeister, 1997). 2. STRUCTURE AND FUNCTION 2.1. Classification and Evolution Cysteine proteases (EC.3.4.22) are proteins of molecular mass about 21-30 kDa. They catalyse the hydrolysis of peptide, amide, ester, thiol ester and thiono ester bonds. The CP family can be subdivided into exopeptidases (e.g. cathepsin X, carboxypeptidase B) and endopeptidases (papain, bromelain, ficain, cathepsins). Exopeptidases cleave the peptide bond proximal to the amino or carboxy termini of the substrate, whereas endopeptidases cleave peptide bonds distant from the N- or C-termini. Cysteine proteases are divided into five clans: CA (papain-like enzymes), 181 J. Polaina and A.P. MacCabe (eds.), Industrial Enzymes, 181–195.
    [Show full text]
  • ^ P X R, for the PURPOSES of INFORMATION ONLY
    WORLD INTELLECTUAL PROPERTY ORGANIZATION PCT International Bureau INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 6 : (11) International Publication Number: WO 98/49190 C07K 5/06, 5/08, 5/10 A l (43) International Publication Date: 5 November 1998 (05.11.98) (21) International Application Number: PCT/US98/08259 (74) Agents: BURKE, John, E. et al.; Cushman Darby & Cushman, Intellectual Property Group of Pillsbury Madison & Sutro, (22) International Filing Date: 24 April 1998 (24.04.98) 1100 New York Avenue, N.W., Washington, DC 20005 (US). (30) Priority Data: 60/044,819 25 April 1997 (25.04.97) US (81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR, Not furnished 23 April 1998 (23.04.98) US BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, GW, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, (71) Applicant (for all designated States except US): CORTECH, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, INC. [US/US]; 6850 North Broadway, Denver, CO 80221 TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW, ARIPO (US). patent (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European (72) Inventors; and patent (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, (75) Inventors/Applicants(for US only): SPRUCE, Lyle, W. IE, IT, LU, MC, NL, PT, SE), OAPI patent (BF, BJ, CF, [US/US]; 948 Camino Del Sol, Chula Vista, CA 91910 CG, Cl, CM, GA, GN, ML, MR, NE, SN, TD, TG).
    [Show full text]
  • Families and Clans of Cysteine Peptidases
    Families and clans of eysteine peptidases Alan J. Barrett* and Neil D. Rawlings Peptidase Laboratory. Department of Immunology, The Babraham Institute, Cambridge CB2 4AT,, UK. Summary The known cysteine peptidases have been classified into 35 sequence families. We argue that these have arisen from at least five separate evolutionary origins, each of which is represented by a set of one or more modern-day families, termed a clan. Clan CA is the largest, containing the papain family, C1, and others with the Cys/His catalytic dyad. Clan CB (His/Cys dyad) contains enzymes from RNA viruses that are distantly related to chymotrypsin. The peptidases of clan CC are also from RNA viruses, but have papain-like Cys/His catalytic sites. Clans CD and CE contain only one family each, those of interleukin-ll3-converting enz3wne and adenovirus L3 proteinase, respectively. A few families cannot yet be assigned to clans. In view of the number of separate origins of enzymes of this type, one should be cautious in generalising about the catalytic mechanisms and other properties of cysteine peptidases as a whole. In contrast, it may be safer to gener- alise for enzymes within a single family or clan. Introduction Peptidases in which the thiol group of a cysteine residue serves as the nucleophile in catalysis are defined as cysteine peptidases. In all the cysteine peptidases discovered so far, the activity depends upon a catalytic dyad, the second member of which is a histidine residue acting as a general base. The majority of cysteine peptidases are endopeptidases, but some act additionally or exclusively as exopeptidases.
    [Show full text]
  • Tesi Dottorato Ilaria Benucci
    Facoltà di Agraria, Dipartimento di Scienze e Tecnologie Agroalimentari (AGR/15) CORSO DI DOTTORATO DI RICERCA Biotecnologia Degli Alimenti - XXIII ciclo Removal of unstable proteins from white wine by immobilized acid protease Coordinatore: Prof. Marco Esti Firma Tutor: Prof. Marco Esti Firma Dottoranda: Ilaria Benucci Firma A Pierluigi ABSTRACT This PhD thesis research project was aimed at assessing and optimizing different immobilization procedures of pineapple stem bromelain, in order to develop an innovative biotechnological technique, alternative to bentonite fining, useful to removal selectively unstable proteins from white wines. Stem bromelain activity was assessed on a suitable synthetic substrate at a reference pH value (3.2), this being the average minimum pH value of wine. Protease was covalently immobilized on different supports by various procedures and the best biocatalyst was chosen measuring immobilization percentage, kinetic parameters and half-life (in model wine buffer). Moreover, the influence on free and immobilized protease activity of potential inhibitors naturally present in wine, such as ethanol, tannins and sulphur dioxide (SO 2) over the average range concentration of wine, was investigated. Finally a kinetic study was carried out using 6 artisan and unrefined white wines, spiced with the synthetic substrate, in order to compare catalytic properties of free and immobilized bromelain. Immobilized protease activity, then, was tested in these wines both on total proteins and on unstable ones. Covalent immobilization reduced bromelain catalytic properties. All kinds of procedures applied at pH 7 allowed the highest immobilization yield. Nevertheless, biocatalysts immobilized at pH 3.2 showed the best catalytic performance. Stem bromelain was successfully immobilized on chitosan beads without glutaraldehyde at pH 3.2, obtaining the most interesting and food-safe biocatalyst, which was used for all other experiments.
    [Show full text]
  • Fortification of Maize Tortilla with an Optimized Chickpea Hydrolysate
    foods Article Fortification of Maize Tortilla with an Optimized Chickpea Hydrolysate and Its Effect on DPPIV Inhibition Capacity and Physicochemical Characteristics Karla A. Acevedo-Martinez and Elvira Gonzalez de Mejia * Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA; [email protected] * Correspondence: [email protected] Abstract: Chickpea hydrolysates have shown bioactivity towards type 2 diabetes by inhibiting dipeptidyl peptidase (DPPIV) activity. The objective was to compare the effect of adding different levels of an optimized bromelain hydrolysate from chickpea isolated protein on DPPIV inhibition capacity and physicochemical properties of maize tortilla. White and blue maize tortillas, with no added chickpea hydrolysates were compared with fortified tortillas at the levels of 5%, 10%, and 15% w/w. Changes in color (L* a* b*, hue angle, and DE), texture (hardness, cohesiveness, and puncture force), and moisture were tested. Soluble protein determination and SDS-PAGE electrophoresis were used to characterize the protein profiles, and LC-MS-MS was used to sequence the peptides. DPPIV inhibition was evaluated before and after simulated gastrointestinal digestion. Peptides in the hydrolysates had high hydrophobicity (7.97–27.05 kcal * mol −1) and pI (5.18–11.13). Molecular docking of peptides showed interaction with DPPIV with an energy of affinity of –5.8 kcal/mol for FDLPAL in comparison with vildagliptin (−6.2 kcal/mol). The lowest fortification level increased Citation: Acevedo-Martinez, K.A.; Gonzalez de Mejia, E. Fortification of soluble protein in 105% (8 g/100 g tortilla). DPPIV inhibition of white maize tortilla increased Maize Tortilla with an Optimized from 11% (fresh control) to 91% (15% fortification), and for blue tortilla from 26% to 95%.
    [Show full text]
  • (12) Patent Application Publication (10) Pub. No.: US 2004/0076614 A1 Schur (43) Pub
    US 2004.0076614A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0076614 A1 Schur (43) Pub. Date: Apr. 22, 2004 (54) SYMBIOTIC REGENERATIVE AGENT Publication Classification (76) Inventor: Jorg PSchur, Wegberg-Dalheim (DE) (51) Int. Cl." ........................... A01N 63/00; A61K 35/76 Correspondence Address: (52) U.S. Cl. ........................................... 424/93.4; 424/780 JENKINS & WILSON, PA 3100 TOWER BLVD SUTE 1400 (57) ABSTRACT DURHAM, NC 27707 (US) The present invention relates to a regenerative medicament for the natural infection-immunological therapy with induc (21) Appl. No.: 10/467,040 tion, regulation and effector mechanisms for the early natu ral cellular immune defense in humans and animals, com (22) PCT Filed: Feb. 1, 2002 prising a special composition with a microorganism (86) PCT No.: PCT/EP02/01056 component and a modulator component, to food, food Supplements, animal feeds and animal feed Supplements (30) Foreign Application Priority Data containing Such composition, and to the use of Such com position for the preparation of regenerative agents for the Feb. 2, 2001 (EP)........................................ O1102.384.3 treatment of humans and animals. US 2004/0076614 A1 Apr. 22, 2004 SYMBIOTC REGENERATIVE AGENT genus Candida”, and R. Hurley/De Louvois report on the “ecological aspects of yeast-like fungi of medical impor 0001. The present invention relates to a regenerative tance”. In Wien. Klin. Wochenschr. 91, 826-830 (1979), O. medicament for the natural infection-immunological therapy Male/Boltz-Nitulescu already described animal trial studies with induction, regulation and effector mechanisms for the on the persorption of Candida albicans and the possibility of early natural cellular immune defense in humans and ani intestinal triggering of an immune response.
    [Show full text]
  • Modulation of Murine Tumor Growth and Colonization by Bromelaine, an Extract of the Pineapple Plant (Ananas Comosum L.)
    in vivo 19: 483-486 (2005) Modulation of Murine Tumor Growth and Colonization by Bromelaine, an Extract of the Pineapple Plant (Ananas Comosum L.) JOSEF BEUTH and JAN MATTHIAS BRAUN Institute for Scientific Evaluation of Naturopathy, University of Koeln, Robert-Koch-Str. 10, 50931 Koeln, Germany Abstract. The antitumor and antimetastatic activities of the endoproteinases trypsin and chymotrypsin and the plant plant cysteine endoproteinase bromelaine were evaluated in a cysteine endoproteinases, bromelaine and papain. Bromelaine murine model. Syngeneic sarcoma L-1 cells were incubated with monotherapy is empirically well established in traumatology, bromelaine (after preceeding time and dosage kinetics) and infectiology and rheumatology. subcutaneously; (s.c.) or intravenously; (i.v.) inoculated into Plant bromelaine is obtained from the stem of the BALB/c-mice (n=5 per experimental group) to induce local pineapple (Ananas comosum L.). Sequencing of the plant tumor growth or lung colonization. Compared to non-protease cysteine endoproteinases, bromelaine and papain, incubated L-1 cells, local tumor growth and experimental lung demonstrated that both are members of the papain family (8). metastasis decreased significantly (p<0.05) after bromelaine Raw stem bromelaine consists of at least 3 immunologically incubation of the tumor cells. Sarcoma L-1 cells induced local distinct proteases: stem bromelaine, fruit bromelaine and tumor growth after s.c. inoculation and lung colonization after ananain (9). High performance liquid cation exchange i.v. injection. Intraperitoneal (i.p.) or s.c. administration of chromatography characterized as many as 9 proteolytically bromelaine (optimal dosage and time schedule tested in active components in raw stem bromelaine (10). Distinct preceeding kinetic studies) significantly (p<0.05) reduced local scientific evaluations are currently being performed to tumor weight, however, lung colonization was non-significantly demonstrate the medical/clinical relevance of those reduced.
    [Show full text]
  • Antibacterial Effects of Proteases on Different Strains of Escherichia Coli and Listeria Monocytogenes Hanan Eshamah Clemson University, [email protected]
    Clemson University TigerPrints All Dissertations Dissertations 8-2013 Antibacterial effects of proteases on different strains of Escherichia coli and Listeria monocytogenes Hanan Eshamah Clemson University, [email protected] Follow this and additional works at: https://tigerprints.clemson.edu/all_dissertations Part of the Food Science Commons Recommended Citation Eshamah, Hanan, "Antibacterial effects of proteases on different strains of Escherichia coli and Listeria monocytogenes" (2013). All Dissertations. 1177. https://tigerprints.clemson.edu/all_dissertations/1177 This Dissertation is brought to you for free and open access by the Dissertations at TigerPrints. It has been accepted for inclusion in All Dissertations by an authorized administrator of TigerPrints. For more information, please contact [email protected]. ANTIBACTERIAL EFFECTS OF PROTEASES ON DIFFERENT STRAINS OF ESCHERICHIA COLI AND LISTERIA MONOCYTOGENS A Dissertation Presented to the Graduate School of Clemson University In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Food Technology by Hanan Lotfi Eshamah August 2013 Accepted by Dr. Paul L. Dawson, Committee Chair Dr. Anthony Pometto III Dr. James Rieck Dr. Xiuping Jiang ABSTRACT Escherichia coli O157:H7 and Listeria monocytogenes are pathogens that have received special attention by federal agencies, food safety researchers and food industries due to their economic and human health impact. To reduce the presence of these pathogens, alternative interventions have been studied. However, increasing consumer’s demand for natural ingredients has made the investigations of effectiveness of natural antimicrobials necessary. In this study, in vitro antimicrobial activity of bromelain and papain against E. coli JM109 and L. monocytogenes was investigated . Furthermore, actinidin and papain were evaluated to reduce populations of L.
    [Show full text]
  • (12) Patent Application Publication (10) Pub. No.: US 2012/0266329 A1 Mathur Et Al
    US 2012026.6329A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2012/0266329 A1 Mathur et al. (43) Pub. Date: Oct. 18, 2012 (54) NUCLEICACIDS AND PROTEINS AND CI2N 9/10 (2006.01) METHODS FOR MAKING AND USING THEMI CI2N 9/24 (2006.01) CI2N 9/02 (2006.01) (75) Inventors: Eric J. Mathur, Carlsbad, CA CI2N 9/06 (2006.01) (US); Cathy Chang, San Marcos, CI2P 2L/02 (2006.01) CA (US) CI2O I/04 (2006.01) CI2N 9/96 (2006.01) (73) Assignee: BP Corporation North America CI2N 5/82 (2006.01) Inc., Houston, TX (US) CI2N 15/53 (2006.01) CI2N IS/54 (2006.01) CI2N 15/57 2006.O1 (22) Filed: Feb. 20, 2012 CI2N IS/60 308: Related U.S. Application Data EN f :08: (62) Division of application No. 1 1/817,403, filed on May AOIH 5/00 (2006.01) 7, 2008, now Pat. No. 8,119,385, filed as application AOIH 5/10 (2006.01) No. PCT/US2006/007642 on Mar. 3, 2006. C07K I4/00 (2006.01) CI2N IS/II (2006.01) (60) Provisional application No. 60/658,984, filed on Mar. AOIH I/06 (2006.01) 4, 2005. CI2N 15/63 (2006.01) Publication Classification (52) U.S. Cl. ................... 800/293; 435/320.1; 435/252.3: 435/325; 435/254.11: 435/254.2:435/348; (51) Int. Cl. 435/419; 435/195; 435/196; 435/198: 435/233; CI2N 15/52 (2006.01) 435/201:435/232; 435/208; 435/227; 435/193; CI2N 15/85 (2006.01) 435/200; 435/189: 435/191: 435/69.1; 435/34; CI2N 5/86 (2006.01) 435/188:536/23.2; 435/468; 800/298; 800/320; CI2N 15/867 (2006.01) 800/317.2: 800/317.4: 800/320.3: 800/306; CI2N 5/864 (2006.01) 800/312 800/320.2: 800/317.3; 800/322; CI2N 5/8 (2006.01) 800/320.1; 530/350, 536/23.1: 800/278; 800/294 CI2N I/2 (2006.01) CI2N 5/10 (2006.01) (57) ABSTRACT CI2N L/15 (2006.01) CI2N I/19 (2006.01) The invention provides polypeptides, including enzymes, CI2N 9/14 (2006.01) structural proteins and binding proteins, polynucleotides CI2N 9/16 (2006.01) encoding these polypeptides, and methods of making and CI2N 9/20 (2006.01) using these polynucleotides and polypeptides.
    [Show full text]
  • Affinity Chromatography As a Key Tool to Purify Protein Protease Inhibitors from Plants
    12 Affinity Chromatography as a Key Tool to Purify Protein Protease Inhibitors from Plants Elizeu Antunes dos Santos, Adeliana Silva de Oliveira, Luciana Maria Araújo Rabêlo, Adriana Ferreira Uchôa and Ana Heloneida Araújo Morais Universidade Federal do Rio Grande do Norte Brazil 1. Introduction Several and distinct physiological processes in all the life forms are dependent on proteases, as processing and turnover of endogenous proteins, digestion of food proteins, regulation of formation and lysis of the clots, activation of apoptosis pathways, plant germination, sporulation, hormone activation, translocation through membranes, fertilization, control of immune response, cell differentiation and growing (Bode & Huber, 2000; Chou & Cai, 2006; Turk et al., 2000). Proteases are also involved in replication and propagation of infectious diseases, and the imbalance of their activity can cause important pathological disorders as inflammation, stroke, cancer and parasite infection (Chou & Cai, 2006; Johansson et al., 2002; Powers et al., 2002). The principal naturally occurring control way of the proteases activity is achieved by the action of inhibitors of protein nature, which bind specifically and block proteases. Protease inhibitors (PIs) are found in all living organisms and are among the most intensively studied proteins. In plants they are widely distributed among different botanical families and have been found in reproductive organs, storage organs and vegetative tissues. They are synthesized constitutively in seeds or can be induced in tissue, as leaves, by the attack of herbivore or abiotic stress (Fan & Wu, 2005; Laskowski & Kato, 1980; Xavier-Filho, 1992). At least four PI families are known and can be distinguished based in their interaction with the protease class that they inhibit (Fan & Wu, 2005; Koiwa et al., 1997; Xavier-Filho, 1992).
    [Show full text]