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(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 22 July 2010 (22.07.2010) WO 2010/083148 Al (51) International Patent Classification: (74) Agents: GAO, Chuan et al.; Townsend and Townsend Cl 2P 21/00 (2006.01) and Crew LLP, Two Embarcadero Center, 8th Floor, San Francisco, California 941 11 (US). (21) International Application Number: PCT/US20 10/020727 (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, (22) International Filing Date: AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, 12 January 2010 (12.01 .2010) CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, (25) Filing Language: English DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, (26) Publication Language: English KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, (30) Priority Data: ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, 61/144,401 13 January 2009 (13.01 .2009) US NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, (71) Applicant (for all designated States except US): SUTRO TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. BIOPHARMA, INC. [US/US]; 310 Utah Street, Suite 150, South San Francisco, California 94080 (US). (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (72) Inventors; and GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, (75) Inventors/Applicants (for US only): VOLOSHIN, Alex- ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, ei M. [US/US]; 3717 1 Sycamore Street, Apt. 137, TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, Newark, California 94560 (US). ZAWADA, James F. ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, [US/US]; 720 Upton Street, Redwood City, California MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, SM, 9406 1 (US). GOLD, Daniel [US/US]; 3223 Santiago TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, Street, San Francisco, California 941 16 (US). ML, MR, NE, SN, TD, TG). [Continued on next page] (54) Title: USE OF DNA GYRASE INHIBITORS FOR IN VITRO POLYPEPTIDE SYNTHESIS REACTIONS (57) Abstract: The present invention provides methods and compositions u se ful for in vitro polypeptide synthesis re Addition of Ciprofloxacin during lysate preparation does not actions. The methods involve the use of impact GM-CSF yields DNA gyrase inhibitors to prevent bacte rial contamination in lysates used for in vitro production of polypeptides. The compositions include contamination-free cell lysates for in vitro protein synthesis reactions. Total E2 Soluble Control Cipro 1µg/mL FIG. 1 Published: USE OF DNA GYRASE INHIBITORS FOR IN VITRO POLYPEPTIDE SYNTHESIS REACTIONS RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Patent Application No. 61/144,401, filed January 13, 2009, the contents of which are incorporated by reference in the entirety for all purposes. FIELD OF THE INVENTION [0002] This invention relates to in vitro polypeptide synthesis. In particular, the invention relates to methods of using antibiotics that are DNA gyrase inhibitors during the in vitro polypeptide synthesis. The invention also provides compositions that are lysates containing DNA gyrase inhibitors for in vitro polypeptide synthesis reactions. The use of DNA gyrase inhibitors during in vitro polypeptide synthesis reactions enhances output of the desired protein. The present invention is particularly useful in an in vitro or cell-free system where protein synthesis is directed by the T7 bacteriophage promoter. BACKGROUND OF THE INVENTION [0003] Protein synthesis is a fundamental biological process that underlies the development of polypeptide therapeutics, vaccines, diagnostics, and industrial enzymes. With the advent of recombinant DNA (rDNA) technology, it has become possible to harness the catalytic machinery of the cell to produce a desired protein. This can be achieved within the cellular environment or in vitro using lysates derived from cells. [0004] In vitro, or cell-free, protein synthesis offers several advantages over conventional in vivo protein expression methods. Cell-free systems can direct most, if not all, of the metabolic resources of the cell towards the exclusive production of one protein. Moreover, the lack of a cell wall and membrane components in vitro is advantageous because it allows for control of the synthesis environment. For example, tRNA levels can be changed to reflect the codon usage of genes being expressed. The redox potential, pH, or ionic strength can also be altered with greater flexibility than with in vivo protein synthesis because concerns of cell growth or viability do not exist. Furthermore, direct recovery of purified, properly folded protein products can be easily achieved. [0005] The productivity of cell-free systems has improved over two orders of magnitude in recent years, from about 5µg/ml-hr to about 150, 250, or 500µg/ml-hr. Such improvement has made in vitro protein synthesis a practical technique for laboratory-scale research and provided a platform technology for high-throughput protein expression. It further indicates the feasibility for using cell-free technologies as an alternative means to in vivo large-scale commercial production of protein pharmaceuticals. [0006] The productivity of in vitro polypeptide synthesis systems can be significantly hindered by bacterial contamination. While many commercially available antibiotics exist for use to inhibit bacterial growth that occurs within an in vitro reaction lysate, most of these antibiotics interfere with protein synthesis or are not acceptable for production of pharmaceuticals. There exists a need for antibiotic application for eliminating bacterial growth in an in vitro polypeptide synthesis reaction lysate without interfering with the in vitro protein synthesis reaction, such that the output of the desired polypeptide is permitted and/or enhanced. The invention described herein fulfills these needs, as will be apparent upon review of the following disclosure. BRIEF SUMMARY OF THE INVENTION [0007] In one aspect, this invention provides a method for in vitro synthesis of a polypeptide. The method includes the step of adding at least one DNA gyrase inhibitor to a polypeptide synthesis reaction lysate in an amount sufficient to inhibit bacterial growth, especially in a reaction lysate where the polypeptide is synthesized from an expression cassette comprising a polynucleotide sequence encoding the polypeptide, where the coding sequence is operably linked to a T7 promoter. The DNA gyrase inhibitor may be a quinolone or an aminocoumarin. [0008] In some embodiments, ciprofloxacin, one of the quinolones, is used in the methods of the present invention. In other embodiments, another quinolone, norfloxacin, is used. Other quinolones useful in the methods of this invention include cinoxacin, flumequine, nalidixic acid, oxolinic acid, piromidic acid, pipemidic acid, rosoxacin, enoxacin, fleroxacin, iomefloxacin, nadifloxacin, ofloxacin, pefloxacin, rufloxacin, balofloxacin, gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin, pazufloxacin, sparfloxacin, temafloxacin, tosufloxacin, clinafloxacin, garenoxacin, gemifloxacin, sitafloxacin, trovafloxacin, prolifloxacin, and ecinofloxacin. [0009] In some embodiments, coumermycin A 1, one of the aminocoumarins, is used in the methods of the present invention. In other embodiments, another aminocoumarin, novobiocin, is used. Clorobiocin is an additional aminocoumarin that may be useful in the cell-free protein synthesis method of this invention. Any combination of two or more of quinolones and/or aminocoumarins, such as those named above, can be used in the method of this invention. [0010] The method of the present invention may be used to suppress the growth of either facilitative anaerobic microorganisms or aerobic microorganisms. [0011] In a second aspect, the present invention provides an in vitro polypeptide synthesis reaction lysate or mixture, which contains at least one DNA gyrase inhibitor in an amount sufficient to inhibit bacterial growth. In particular, this reaction mixture contains an expression cassette that includes a polynucleotide sequence encoding a polypeptide to be synthesized, operably linked to a T7 promoter. The DNA gyrase inhibitor may be a quinolone or an aminocoumarin. [0012] In some embodiments, ciprofloxacin, one of the quinolones, is used in the reaction mixtures of the present invention. In other embodiments, another quinolone, norfloxacin, is used. Other quinolones useful in the reaction mixtures of this invention include cinoxacin, flumequine, nalidixic acid, oxolinic acid, piromidic acid, pipemidic acid, rosoxacin, enoxacin, fleroxacin, iomefloxacin, nadifloxacin, ofloxacin, pefloxacin, rufloxacin, balofloxacin, gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin, pazufloxacin, sparfloxacin, temafloxacin, tosufloxacin, clinafloxacin, garenoxacin, gemifloxacin, sitafloxacin, trovafloxacin, prolifloxacin, and ecinofloxacin. [0013] In some embodiments, coumermycin A 1, one of the aminocoumarins, is used in the reaction mixtures of the present invention. In other embodiments, another aminocoumarin, novobiocin, is used. Clorobiocin is an additional aminocoumarin that may be useful in the cell-free protein synthesis reaction lysate of this invention. Any combination of
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  • Molecular and Immunological Sd0100025 Characterization of Mycobacteria Associated with Bovine Farcy

    Molecular and Immunological Sd0100025 Characterization of Mycobacteria Associated with Bovine Farcy

    MOLECULAR AND IMMUNOLOGICAL SD0100025 CHARACTERIZATION OF MYCOBACTERIA ASSOCIATED WITH BOVINE FARCY. Thesis submitted in accordance with requirements of the University of Khartoum for the Degree of Doctor of Philosophy (Ph.D). By Victor Loku Kwajok B.V.Med. 1978, University of Cairo/Egypt. M.V.Sc.1992, University of Khartoum/Sudan. Supervisor: Dr. Maowia M. Mukhtar. Institute of Endemic Diseases, University of Khartoum. Department of Preventive Medicine and Veterinary Public Health. Faculty of Veterinary Science, University of Khartoum. 2000. 32/27 SOME PAGES ARE MISSING IN THE ORIGINAL DOCUMENT LIST OF CONTENTS DEDICATION. * ii v ACKNOWLEDGEMENTS vi ABBREVIATIONS viii ABSTRACT. xi CHAPTER ONE. REVIEW OF LITERATURE. 1. General Introduction. 1. 1.1. Molecular Systematics of genus Mycobacterium. 2. 1.2. Molecular taxonomy of M. farcinogenese and M. senegalense 11. 1.3. Immunology of bovine farcy agents. 36. CHAPTER TWO. MATERIALS AND METHODOLOGY 41. Isolation, identification and characterization of M. farcinogenes. 2.1 Phenotypic characterization. 41. 2.1.1. Morphological and biochemical tests. 2.1.2. Degradation tests. 2.1.3. Rapid fluorogenic enzyme tests. 2.1.4. Nutritional tests. 2.1.5. Physiological tests. 2.2. Molecular characterization. 52. 2.2.1. DNA extraction and purification 2.2.2. PCR amplification and application. 2.2.3. DNA sequencing of 16SrDNA. 2.2.4. PCR-based restriction fragment length polymorphism. 2.3 Imrmmological analyses of bovine farcy agents. 71. 2.3.1 .EL1SA technique for sera diagnosis. 2.3.2 Animal pathogenicity Tests. 2.3.3 Protein antigen profiles determination using. CHAPTER THREE: RESULTS. 78. CHAPTER. FOUR: DISCUSSION. 124. REFERENCES. 133.