Name: a high yield and cost-efficient expression system of human granzymes in mammalian cells
Uploaded: 2015-06-10T15:00:00
Description: Watch this Scientific Journal Video about A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells at JoVE.com

This work was supported by grants from the Novartis Foundation for Medical-Biological Research and from the Research Pool of the University of Fribourg (to MW). We thank Li Zhao, Zhan Xu, and Solange Kharoubi Hess for technical support, as well as Radu Aricescu and Yvonne Jones (Oxford University, UK) for providing the pHLsec plasmid, and Thomas Sch&#252;rpf (Harvard Medical School) for helpful discussions.

अभिव्यक्ति प्लाज्मिड pHLsec-Gzm की 1. उत्पादन <ol><li> मानव एन.के. कोशिकाओं से manufacturer` के बाद एक उपयुक्त आरएनए अलगाव विधि का उपयोग कर और एक प्रथम कतरा सीडीएनए synthesize किट का उपयोग कर टाइप रिवर्स (26 या सभी पांच मानव Gzms व्...

<ol>
	<li>Chowdhury, D., Lieberman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Death+by+a+thousand+cuts:+granzyme+pathways+of+programmed+cell+death.">Death by a thousand cuts: granzyme pathways of programmed cell death.</a> Annu Rev Immunol. 26, 389-420 (2008).</li><li>Thiery, J., Lieberman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perforin:+a+key+pore-forming+protein+for+immune+control+of+viruses+and+cancer.">Perforin: a key pore-forming protein for immune control of viruses and cancer.</a> Subcell Biochem. 80, 197-220 (2014).</li><li>Krensky, A. M., Clayberger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+and+clinical+relevance+of+granulysin.">Biology and clinical relevance of granulysin.</a> Tissue Antigens. 73, 193-198 (2009).</li><li>Bovenschen, N., Kummer, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orphan+granzymes+find+a+home.">Orphan granzymes find a home.</a> Immunol Rev. 235, 117-127 (2010).</li><li>Lieberman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granzyme+A+activates+another+way+to+die.">Granzyme A activates another way to die.</a> Immunol Rev. 235, 93-104 (2010).</li><li>Ewen, C. L., Kane, K. P., Bleackley, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quarter+century+of+granzymes.">A quarter century of granzymes.</a> Cell death and differentiation. 19, 28-35 (2012).</li><li>Hiebert, P. R., Granville, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granzyme+B+in+injury,+inflammation,+and+repair.">Granzyme B in injury, inflammation, and repair.</a> Trends Mol Med. 18, 732-741 (2012).</li><li>Afonina, I. S., Cullen, S. P., Martin, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+and+non-cytotoxic+roles+of+the+CTL/NK+protease+granzyme.">Cytotoxic and non-cytotoxic roles of the CTL/NK protease granzyme.</a> B. Immunol Rev. 235, 105-116 (2010).</li><li>Walch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+cells+kill+intracellular+bacteria+through+granulysin-mediated+delivery+of+granzymes.">Cytotoxic cells kill intracellular bacteria through granulysin-mediated delivery of granzymes.</a> Cell. 157, 1309-1323 (2014).</li><li>Lee, W. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invariant+natural+killer+T+cells+act+as+an+extravascular+cytotoxic+barrier+for+joint-invading+Lyme+Borrelia.">Invariant natural killer T cells act as an extravascular cytotoxic barrier for joint-invading Lyme Borrelia.</a> Proc Natl Acad Sci U S A. , (2014).</li><li>Kapelski, S., de Almeida, M., Fischer, R., Barth, S., Fendel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimalarial+activity+of+granzyme+B+and+its+targeted+delivery+by+a+granzyme+B-scFv+fusion+protein.">Antimalarial activity of granzyme B and its targeted delivery by a granzyme B-scFv fusion protein.</a> Antimicrob Agents Chemother. , (2014).</li><li>Thiery, J., Walch, M., Jensen, D. K., Martinvalet, D., Lieberman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+cytotoxic+T+cell+and+NK+granules+and+purification+of+their+effector+proteins.">Isolation of cytotoxic T cell and NK granules and purification of their effector proteins.</a> Curr Protoc Cell Biol. Chapter 3 (Unit3 37), (2010).</li><li>Shi, L., Yang, X., Froelich, C. J., Greenberg, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+use+of+granzyme+B.">Purification and use of granzyme B.</a> Methods Enzymol. 322, 125-143 (2000).</li><li>Masson, D., Tschopp, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+family+of+serine+esterases+in+lytic+granules+of+cytolytic+T+lymphocytes.">A family of serine esterases in lytic granules of cytolytic T lymphocytes.</a> Cell. 49, 679-685 (1987).</li><li>Lorentsen, R. H., Fynbo, C. H., Thogersen, H. C., Etzerodt, M., Holtet, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression,+refolding,+and+purification+of+recombinant+human+granzyme+B.">Expression, refolding, and purification of recombinant human granzyme B.</a> Protein Expr Purif. 39, 18-26 (2005).</li><li>Sun, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+purification+of+recombinant+human+granzyme+B+from+Pichia+pastoris.">Expression and purification of recombinant human granzyme B from Pichia pastoris.</a> Biochem Biophys Res Commun. 261, 251-255 (1999).</li><li>Xia, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+purification+of+enzymatically+active+recombinant+granzyme+B+in+a+baculovirus+system.">Expression and purification of enzymatically active recombinant granzyme B in a baculovirus system.</a> Biochem Biophys Res Commun. 243, 384-389 (1998).</li><li>Stahnke, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granzyme+B-H22(scFv),+a+human+immunotoxin+targeting+CD64+in+acute+myeloid+leukemia+of+monocytic+subtypes.">Granzyme B-H22(scFv), a human immunotoxin targeting CD64 in acute myeloid leukemia of monocytic subtypes.</a> Mol Cancer Ther. 7, 2924-2932 (2008).</li><li>Gehrmann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+expression+and+purification+system+for+the+production+of+enzymatic+and+biologically+active+human+granzyme.">A novel expression and purification system for the production of enzymatic and biologically active human granzyme.</a> B. J Immunol Methods. 371, 8-17 (2011).</li><li>Metkar, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+cell+granule-mediated+apoptosis:+perforin+delivers+granzyme+B-serglycin+complexes+into+target+cells+without+plasma+membrane+pore+formation.">Cytotoxic cell granule-mediated apoptosis: perforin delivers granzyme B-serglycin complexes into target cells without plasma membrane pore formation.</a> Immunity. 16, 417-428 (2002).</li><li>Motyka, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mannose+6-phosphate/insulin-like+growth+factor+II+receptor+is+a+death+receptor+for+granzyme+B+during+cytotoxic+T+cell-induced+apoptosis.">Mannose 6-phosphate/insulin-like growth factor II receptor is a death receptor for granzyme B during cytotoxic T cell-induced apoptosis.</a> Cell. 103, 491-500 (2000).</li><li>Pinkoski, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+and+trafficking+of+granzyme+B+in+target+cells+during+granzyme+B-perforin-mediated+apoptosis.">Entry and trafficking of granzyme B in target cells during granzyme B-perforin-mediated apoptosis.</a> Blood. 92, 1044-1054 (1998).</li><li>Aricescu, A. R., Lu, W., Jones, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+time-+and+cost-efficient+system+for+high-level+protein+production+in+mammalian+cells.">A time- and cost-efficient system for high-level protein production in mammalian cells.</a> Acta Crystallogr D Biol Crystallogr. 62, 1243-1250 (2006).</li><li>Fukuchi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+assays+of+nine+heterogeneous+promoters+in+neural+and+other+cultured+cells.">Activity assays of nine heterogeneous promoters in neural and other cultured cells.</a> In Vitro Cell Dev Biol Anim. 30 A, 300-305 (1994).</li><li>Tran, T. V., Ellis, K. A., Kam, C. M., Hudig, D., Powers, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dipeptidyl+peptidase+I:+importance+of+progranzyme+activation+sequences,+other+dipeptide+sequences,+and+the+N-terminal+amino+group+of+synthetic+substrates+for+enzyme+activity.">Dipeptidyl peptidase I: importance of progranzyme activation sequences, other dipeptide sequences, and the N-terminal amino group of synthetic substrates for enzyme activity.</a> Arch Biochem Biophys. 403, 160-170 (2002).</li><li>Somanchi, S. S., Senyukov, V. V., Denman, C. J., Lee, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion,+purification,+and+functional+assessment+of+human+peripheral+blood+NK+cells.">Expansion, purification, and functional assessment of human peripheral blood NK cells.</a> Journal of visualized experiments : JoVE. , (2011).</li><li>Barry, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granzyme+B+short-circuits+the+need+for+caspase+8+activity+during+granule-mediated+cytotoxic+T-lymphocyte+killing+by+directly+cleaving+Bid.">Granzyme B short-circuits the need for caspase 8 activity during granule-mediated cytotoxic T-lymphocyte killing by directly cleaving Bid.</a> Mol Cell Biol. 20, 3781-3794 (2000).</li><li>Darmon, A. J., Nicholson, D. W., Bleackley, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+apoptotic+protease+CPP32+by+cytotoxic+T-cell-derived+granzyme+B.+.">Activation of the apoptotic protease CPP32 by cytotoxic T-cell-derived granzyme B. .</a> Nature. 377, 446-448 (1995).</li><li>Rajani, D. K., Walch, M., Martinvalet, D., Thomas, M. P., Lieberman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+RNA+processing+during+immune-mediated+programmed+cell+death.">Alterations in RNA processing during immune-mediated programmed cell death.</a> Proc Natl Acad Sci U S A. 109, 8688-8693 (2012).</li><li>Domselaar, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noncytotoxic+inhibition+of+cytomegalovirus+replication+through+NK+cell+protease+granzyme+M-mediated+cleavage+of+viral+phosphoprotein+71.">Noncytotoxic inhibition of cytomegalovirus replication through NK cell protease granzyme M-mediated cleavage of viral phosphoprotein 71.</a> J Immunol. 185, 7605-7613 (2010).</li><li>Thiery, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perforin+activates+clathrin-+and+dynamin-dependent+endocytosis,+which+is+required+for+plasma+membrane+repair+and+delivery+of+granzyme+B+for+granzyme-mediated+apoptosis.">Perforin activates clathrin- and dynamin-dependent endocytosis, which is required for plasma membrane repair and delivery of granzyme B for granzyme-mediated apoptosis.</a> Blood. 115, 1582-1593 (2010).</li><li>Thiery, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perforin+pores+in+the+endosomal+membrane+trigger+the+release+of+endocytosed+granzyme+B+into+the+cytosol+of+target+cells.">Perforin pores in the endosomal membrane trigger the release of endocytosed granzyme B into the cytosol of target cells.</a> Nat Immunol. 12, 770-777 (2011).</li><li>Thomas, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+protease+binding+to+nucleic+acids+promotes+nuclear+localization+and+cleavage+of+nucleic+acid+binding+proteins.">Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic acid binding proteins.</a> J Immunol. 192, 5390-5397 (2014).</li><li>Jacquemin, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granzyme+B-induced+mitochondrial+ROS+are+required+for+apoptosis.">Granzyme B-induced mitochondrial ROS are required for apoptosis.</a> Cell death and differentiation. , (2014).</li><li>Kummer, J. A., Kamp, A. M., Citarella, F., Horrevoets, A. J., Hack, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+human+recombinant+granzyme+A+zymogen+and+its+activation+by+the+cysteine+proteinase+cathepsin+C.">Expression of human recombinant granzyme A zymogen and its activation by the cysteine proteinase cathepsin C.</a> The Journal of biological chemistry. 271, 9281-9286 (1996).</li><li>Hagn, M., Sutton, V. R., Trapani, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+colorimetric+assay+that+specifically+measures+Granzyme+B+proteolytic+activity:+hydrolysis+of+Boc-Ala-Ala-Asp-S-Bzl.">A colorimetric assay that specifically measures Granzyme B proteolytic activity: hydrolysis of Boc-Ala-Ala-Asp-S-Bzl.</a> Journal of visualized experiments : JoVE. , e52419 (2014).</li><li>Huang, M. T., Gorman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intervening+sequences+increase+efficiency+of+RNA+3'+processing+and+accumulation+of+cytoplasmic+RNA.">Intervening sequences increase efficiency of RNA 3' processing and accumulation of cytoplasmic RNA.</a> Nucleic Acids Res. 18, 937-947 (1990).</li><li>Luthman, H., Magnusson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency+polyoma+DNA+transfection+of+chloroquine+treated+cells.">High efficiency polyoma DNA transfection of chloroquine treated cells.</a> Nucleic Acids Res. 11, 1295-1308 (1983).</li><li>Magee, A. I., Grant, D. A., Hermon-Taylor, J., Offord, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+one-stage+method+for+assay+of+enterokinase+activity+by+release+of+radiolabelled+activation+peptides+from+alpha-N-[3H]acetyl-trypsinogen+and+the+effect+of+calcium+ions+on+the+enzyme+activity.">Specific one-stage method for assay of enterokinase activity by release of radiolabelled activation peptides from alpha-N-[3H]acetyl-trypsinogen and the effect of calcium ions on the enzyme activity.</a> Biochem J. 197, 239-244 (1981).</li></ol>

GzmA और GzmB के शास्त्रीय और बड़े पैमाने पर अध्ययन भूमिका ताकना के गठन प्रोटीन PFN 1 से उनके साइटोसोलिक प्रसव के बाद स्तनधारी कोशिकाओं में apoptosis के शामिल है। हाल ही में, Gzms की साइटोटोक्सिक स्पेक्ट्रम बैक्टीर?...

Gzms विशेष सीटीएल की लाइसोसोम और एन.के. कोशिकाओं 1 में स्थानीय अत्यधिक मुताबिक़ सेरीन प्रोटिएजों के एक परिवार के हैं। इन हत्यारा कोशिकाओं की साइटोटोक्सिक कणिकाओं भी उन्मूलन 2,3 के लिए किस्मत में एक लक्ष्य सेल की मान्यता पर Gzms के साथ एक साथ जारी कर रहे हैं कि झिल्ली में खलल न डालें प्रोटीन PFN और GNLY होते हैं। (- जी, कश्मीर, और एम एन GzmA) मानव (GzmA, बी, एच, कश्मीर और एम) में पांच Gzms, और चूहों में 10 Gzms कर रहे हैं। GzmA और GzmB सबसे प्रचुर मात्रा में है और बड़े पैमाने पर इंसानों और चूहों 1 में अध्ययन कर रहे हैं। हालाँकि, और अधिक हाल ही के अध्ययन सेल मौत रास्ते के रूप में अच्छी तरह से स्वास्थ्य और रोग 4 में अन्य तथाकथित अनाथ Gzms द्वारा मध्यस्थता अतिरिक्त जैविक प्रभाव की जांच करने के लिए शुरू कर दिया है। PFN 5,6 द्वारा लक्ष्य कोशिकाओं में कर दिया जब Gzms का सबसे अच्छा ज्ञात समारोह, GzmA और GzmB की विशेष रूप से, स्तनधारी कोशिकाओं में क्रमादेशित कोशिका मृत्यु के शामिल है। लेकिन, और अधिक हाल के अध्ययनों से यह भी PFN 7,8 द्वारा स्वतंत्र रूप से साइटोसोलिक प्रसव की, प्रतिरक्षा विनियमन और सूजन पर गहरा प्रभाव के साथ Gzms के बाह्य प्रभावों का प्रदर्शन किया। Gzms के साइटोसोलिक प्रवेश के बाद कुशलता से मारे गए हैं कि कोशिकाओं के स्पेक्ट्रम भी हाल ही में बैक्टीरिया 9,10 और यहां तक कि कुछ परजीवी से 11 स्तनधारी कोशिकाओं से चौड़ी हो गई थी। ये हाल की खोजों Gzm शोधकर्ताओं के लिए एक नया क्षेत्र खोल दिया। इसलिए, एक लागत प्रभावी, उच्च उपज स्तनधारी अभिव्यक्ति प्रणाली काफी उन भविष्य के अध्ययन के लिए रास्ता आसान हो जाएगा। मूल निवासी मानव, माउस और चूहे Gzms सफलतापूर्वक सीटीएल और एन.के. कोशिकाओं लाइनों 12-14 का दाना अंश से शुद्ध किया गया है। हालांकि, हमारे हाथ में इस तरह के शोधन तकनीक की उपज कम से कम 0.1 मिलीग्राम / एल सेल संस्कृति (अप्रकाशित अवलोकन और 12) की सीमा में है। इसके अलावा, othe के द्वारा संक्रमण के बिना एक भी Gzm की chromatographic संकल्पआर Gzms और / या कणिकाओं में भी मौजूद हैं कि प्रोटीन (अप्रकाशित डेटा और 12,14) चुनौती दे रहा है। संयोजक Gzms भी इस तरह के HEK 293 18,19 के रूप में स्तनधारी कोशिकाओं में बैक्टीरिया 15, खमीर 16, कीट कोशिकाओं 17 में और में तैयार किए गए। केवल स्तनधारी अभिव्यक्ति सिस्टम देशी साइटोटोक्सिक प्रोटीन के समान posttranslational संशोधनों के साथ पुनः संयोजक एंजाइमों का उत्पादन करने की क्षमता सहन। Posttranslational संशोधनों endocytosis के द्वारा विशिष्ट तेज और लक्ष्य कोशिकाओं 20-22 भीतर प्रोटीज के intracellular स्थानीयकरण के साथ फंसाया गया है। इसलिए, pHLsec 23 का उपयोग करके Gzm अभिव्यक्ति के लिए प्लाज्मिड रीढ़ के रूप में (राडू Aricescu और वोन जोन्स, ऑक्सफोर्ड, ब्रिटेन के विश्वविद्यालय के एक तरह का उपहार), हम में उच्च उपज प्रोटीन के उत्पादन के लिए एक सरल, समय और लागत प्रभावी प्रणाली की स्थापना HEK293T कोशिकाओं। pHLsec एक चिकन Β actin के प्रमोटर के साथ एक सीएमवी बढ़ाने को जोड़ती है; साथ में, इन तत्वों प्रदर्शन हैटेड विभिन्न सेल लाइनों 24 में सबसे मजबूत प्रमोटर गतिविधि। इसके अलावा, प्लाज्मिड एक खरगोश Β ग्लोबिन Intron, अनुकूलित कोज़ाक और स्राव का संकेत है, एक लिस-6xHis-टैग और एक पाली-एक संकेत होता है। इंसर्ट के स्राव संकेत और उपयुक्त एन टर्मिनल डोमेन के कमी प्रोटीन के लिए इष्टतम अभिव्यक्ति और स्राव दक्षता सुनिश्चित करने लिस-6xHis-टैग (चित्रा 1) के बीच आसानी से क्लोन किया जा सकता है। Gzms की अभिव्यक्ति के लिए, हम इतना है कि ई.के. उपचार स्रावित Gzms (सक्रिय Gzms एन टर्मिनल के साथ शुरू सक्रिय एक enterokinase (ई.के.) साइट (DDDDK) द्वारा पीछा वेक्टर द्वारा प्रदान की गई स्राव संकेत के साथ अंतर्जात स्राव संकेत अनुक्रम की जगह अमीनो एसिड अनुक्रम IIGG 25)। इसके साथ ही इस विधि के लिए पक्ष में, HEK293T कोशिकाओं ऐसे Dulbecco संशोधित ईगल मध्यम (DMEM) के रूप में, कम कीमत माध्यम में तेजी से बढ़ती है, और लागत प्रभावी कैल्शियम फॉस्फेट अभिकर्मक विधि के लिए अच्छी तरह से अनुकूल हैं।

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Invitrogen</td>
			<td>15596-026</td>
			<td>Total RNA isolation kit</td>
		</tr>
		<tr>
			<td>SuperScript II Reverse Transcriptase</td>
			<td>Invitrogen</td>
			<td>18064-014</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>NEB</td>
			<td>M0530L</td>
			<td></td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit</td>
			<td>QIAGEN</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr>
			<td>EX-CELL 293 Serum-Free Medium for HEK 293 Cells</td>
			<td>Sigma</td>
			<td>14571C</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, GlutaMAX Supplement, pyruvate</td>
			<td>Gibco</td>
			<td>31966-021</td>
			<td></td>
		</tr>
		<tr>
			<td>SnakeSkin Dialysis Tubing, 10K MWCO</td>
			<td>Thermo Scientific</td>
			<td>68100</td>
			<td></td>
		</tr>
		<tr>
			<td>Enterokinase from bovine intestine</td>
			<td>Sigma</td>
			<td>E4906</td>
			<td>recombinant, &#8805;20&#160;units/mg protein</td>
		</tr>
		<tr>
			<td>HisTrap Excel 5ml-column</td>
			<td>GE Healthcare</td>
			<td>17-3712-06&#160;</td>
			<td>Nickel IMAC</td>
		</tr>
		<tr>
			<td>HiTrap SP HP 5 ml-column</td>
			<td>GE Healthcare</td>
			<td>17-1152-01&#160;</td>
			<td>S column</td>
		</tr>
		<tr>
			<td>N-&#945;-Cbz-L-lysine thiobenzylester (BLT)</td>
			<td>Sigma</td>
			<td>C3647</td>
			<td>GzmA substrate</td>
		</tr>
		<tr>
			<td>Boc-Ala-Ala-Asp-S-Bzl (AAD)</td>
			<td>MP Biomedicals</td>
			<td>2193608 _10mg</td>
			<td>GzmB substrate</td>
		</tr>
		<tr>
			<td>Suc-Ala-Ala-Pro-Leu-p-nitroanilide (AAPL)</td>
			<td>Bachem</td>
			<td></td>
			<td>GzmM substrate</td>
		</tr>
		<tr>
			<td>5,5&#8242;-dithio-bis(2-nitrobenzoic acid) (DTNB)</td>
			<td>Sigma</td>
			<td>D8130</td>
			<td>Ellman`s reagent</td>
		</tr>
	</tbody>
</table>

a high yield and cost-efficient expression system of human granzymes in mammalian cells

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their cytotoxic granule content to eliminate the target cells and the intracellular pathogen. Death of the host cells and intracellular pathogens is triggered by the granule serine proteases, granzymes (Gzms), delivered into the host cell cytosol by the pore forming protein perforin (PFN) and into bacterial pathogens by the prokaryotic membrane disrupting protein granulysin (GNLY). To investigate the molecular mechanisms of target cell death mediated by the Gzms in experimental in-vitro settings, protein expression and purification systems that produce high amounts of active enzymes are necessary. Mammalian secreted protein expression systems imply the potential to produce correctly folded, fully functional protein that bears posttranslational modification, such as glycosylation. Therefore, we used a cost-efficient calcium precipitation method for transient transfection of HEK293T cells with human Gzms cloned into the expression plasmid pHLsec. Gzm purification from the culture supernatant was achieved by immobilized nickel affinity chromatography using the C-terminal polyhistidine tag provided by the vector. The insertion of an enterokinase site at the N-terminus of the protein allowed the generation of active protease that was finally purified by cation exchange chromatography. The system was tested by producing high levels of cytotoxic human Gzm A, B and M and should be capable to produce virtually every enzyme in the human body in high yields.

निम्नलिखित खंड में हम विधि रोशन करने के लिए एक GzmA तैयारी की एक पूरी प्रलेखन पेश करेंगे। हम भी सफलतापूर्वक शोधन क्षमता और गतिविधि के संबंध में इसी तरह के परिणाम का उत्पादन, GzmB और GzmM शुद्ध। हालांकि, उन बाद म?...

Watch this Scientific Journal Video about A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells at JoVE.com

A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease.

स्तनधारी कोशिकाओं में मानव Granzymes की एक उच्च उपज और लागत प्रभावी अभिव्यक्ति प्रणाली

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their ...

Research

JoVE Journal

Biology

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

जीन Pulser MXcell electroporation प्रणाली का उपयोग करने के लिए उच्च क्षमता के साथ प्राथमिक कोशिकाओं transfect

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

एक स्वचालित सेल काउंटर का उपयोग करने के लिए जीन एक्सप्रेशन अध्ययन सरल: आईएल-4 निर्भर जीन एक्सप्रेशन के Namalwa कक्ष में siRNA नॉकडाउन

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

Chromatin परख सक्रिय मानव कोशिकाओं में जीन एक्सप्रेशन में गतिशील histone संशोधन करने के लिए immunoprecipitation (चिप)

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

Absolute Quantum Yield Measurement of Powder Samples

पाउडर नमूने के पूर्ण क्वांटम यील्ड मापन

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

लघु अणु प्रेरण के साथ pluripotent मानव भ्रूण स्टेम कोशिकाओं से मानव कार्डिएक व्यापारियों और cardiomyocytes के कुशल व्युत्पत्ति

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Analysis of Cell Cycle Position in Mammalian Cells

सेल चक्र स्थिति के स्तनधारी कोशिकाओं में विश्लेषण

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Expression Analysis of Mammalian Linker-histone Subtypes

स्तनधारी Linker histone subtypes की अभिव्यक्ति विश्लेषण

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

स्तनधारी कोशिकाओं में endoplasmic जालिका स्थानीयकृत mRNAs का विज़ुअलाइज़ेशन

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

संवर्धित स्तनधारी कोशिकाओं में जैविक रास्ते की Ubiquitin और ubiquitin की तरह सिस्टम नियामकों की पहचान siRNA स्क्रीनिंग

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

कुशल स्तनधारी सेल अभिव्यक्ति और क्रिस्टलीकरण के लिए कोशिकी glycoproteins की एकल कदम शुद्धीकरण

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...