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.

발현 플라스미드 pHLsec-GZM 1. 생산 <ol><li> 인간 NK 세포로부터 manufacturer` 다음 적합한 RNA 분리 방법을 이용하여 첫 번째 가닥의 cDNA를 합성 키트를 사용하여 스크립트 작성 역방향 (26 또는 다섯 인간 Gzms 발현 NK 세포주 NK-92에서와 같이 제조 차 전지), 총 RNA를 준비 의 권장 사항. 23 (라두 Aricescu과 이본 존스, 옥스포드, 영국의 대학 종류의 선물)에 설명 된대로 GZM cDNA를 AgeI과 KpnI으로 ?...

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	<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 및 GzmB의 고전과 광범위하게 연구 역할은 기공 형성 단백질 PFN 1로 그들의 세포질 배달 후 포유 동물 세포에서 세포 사멸의 유도이다. 최근 Gzms의 세포 독성 스펙트럼 세균 9,10뿐만 아니라 특정 기생충 (11)에 포유류 세포에서 현저하게 확대시켰다. 또한, GzmA 및 GzmB의 비 고전적, 세포 외 기능뿐만 아니라 다양한 고아 Gzms의 생물학적 중요성은 여전히​​ 불분명하다. 이 ...

Gzms 전문 CTL의 리소좀과 NK 세포 1 지역화 높은 상동 세린 프로테아제의 가족입니다. 이러한 킬러 세포의 세포 독성 과립은 제거 2,3 향하는 표적 세포의 인식에 Gzms와 동시에 출시되는 막 방해 단백질 PFN 및 GNLY가 포함되어 있습니다. (- G, K, M과 N GzmA) 인간 (GzmA, B, H, K와 M)에서 다섯 Gzms 및 마우스에서 10 Gzms이 있습니다. GzmA과 GzmB이 가장 풍부하고 광범위하게 인간과 생쥐 한 연구이다. 그러나, 최근의 연구들은 세포 사멸 경로뿐만 아니라 건강과 질환 4 다른, 소위 고아 Gzms에 의해 매개되는 생물학적 효과를 추가로 조사하기 시작했다. 5,6- PFN에 의해 표적 세포 내로 전달 된 경우 Gzms의 가장 잘 알려진 함수와 GzmA GzmB의 특히 포유 동물 세포에서 프로그래밍 된 세포 사멸을 유도한다. 그러나더 최근의 연구는 또한 PFN 7,8 독립적으로 세포질 전달, 면역 조절 및 염증에 깊은 영향을 Gzms의 세포 외 효과를 보여 주었다. Gzms의 세포질 기입 한 후 효율적으로 살해 세포의 스펙트럼은 최근 박테리아 9, 10, 심지어 특정 기생충 (11) 포유 동물 세포에서 확대되었다. 이러한 최근의 발견은 GZM 연구원에 대한 완전히 새로운 분야를 열었다. 따라서, 비용 효율적인, 고 수율 포유 동물 발현 시스템은 크게 그 미래 연구에 대한 방법을 완화합니다. 네이티브 인간, 마우스 및 래트 Gzms 성공적 CTL 및 NK 세포 라인 12-14의 과립 분획으로부터 정제 하였다. 그러나, 우리의 손에 이러한 정제 기술의 수율은 0.1 ㎎ / ℓ, 세포 배양 (미공개 관측 및 12)의 범위이다. 또한, 인접하여 오염이없는 단일의 GZM 크로마토 해상도R Gzms 및 / 또는 과립 형태로도 존재하는 단백질 (게시되지 않은 데이터와 12, 14)에 도전하고있다. 재조합 Gzms 심지어 이러한 HEK 293 18,19 같은 포유 동물 세포에서 세균 15, 16, 효모, 곤충 세포 및 17에서 제조 하였다. 만 포유류 발현 시스템은 원시 세포 독성 단백질과 동일한 번역 후 변형으로 재조합 효소를 생산할 수있는 가능성을 부담. 번역 후 변형은 엔도 사이토 시스에 의해 특정 흡수 및 표적 세포 20-22 내의 단백질 분해 효소의 세포 내 현지화에 연루되어있다. 따라서, pHLsec (23)를 사용하여 GZM 발현 플라스미드 백본 (라두 Aricescu과 이본 존스, 옥스포드, 영국의 대학의 일종 선물), 우리는 높은 수율 단백질 생산을위한 간단하고, 시간과 비용 효율적인 시스템을 구축 HEK293T 세포. pHLsec는 닭 Β-액틴 프로모터와 CMV 증강을 결합; 함께, 이러한 요소는 demonstra테드 다양한 세포 라인 (24)에서 가장 강력한 프로모터 활성. 또한, 플라스미드는 토끼 Β-글로빈 인트론, 최적화 된 코작과 분비 신호리스 - 6xHis 태그 및 폴리 신호가 포함되어 있습니다. 삽입이 분비 신호와 적절한 N- 말단 도메인을 부족한 단백질에 대한 최적의 발현과 분비 효율을 보장리스 - 6xHis 태그 (그림 1) 사이에 편리하게 복제 할 수 있습니다. Gzms의 발현을 위해, 우리는되도록 EK 치료 분비 Gzms는 (활성 Gzms는 N - 말단으로 시작 활성화 된 엔테로 키나제 (EK) 사이트 (DDDDK) 뒤에 벡터에 의해 제공 분비 신호 내인성 분비 신호 서열로 대체 아미노산 서열 IIGG 25). 또한이 방법에 대한 찬성, HEK293T 세포는 둘 베코의 수정 이글의 중간 (DMEM)로, 저가의 매체에 빠른 속도로 성장하고, 비용 효율적인 칼슘 포스페이트 형질 전환 방법에 적합하다.

<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.

다음 섹션에서 우리는 방법을 조명 GzmA 준비의 전체​​ 문서를 발표 할 예정이다. 또한 성공적으로 정화 효율 및 작동에 대해서는 유사한 결과를 생성하고 GzmB GzmM 정제. 그러나, 그 후 준비에서 우리는 데이터의 몇 선택한 조각을 보여줍니다. 현재 프로토콜 다음 293T 세포로부터 GzmB 제제는 다양한 생물학적 분석 시스템 9,29,31-34 그들의 활동을 강조 여러 발표 된 연구에 사용 하였다. <p...

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.

포유 동물 세포에서 인간의 Granzymes의 높은 수율과 비용 효율적인 발현 시스템

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

고효율 기본 셀을 Transfect하는 진 Pulser Electroporation MXcell의 시스템을 사용하여

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

연구

생물학

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

유전자 발현 연구를 단순화하는 자동 셀 카운터를 사용하여 : Namalwa 셀에서 IL - 4 종속 유전자 발현의 siRNA의 최저

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

인간의 세포에서 활성 유전자 발현의 어세 동적 히스톤 수정을 염색질 Immunoprecipitation (칩)

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

파우더 샘플의 절대 양자 수율 측정

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

작은 분자와 유도 Pluripotent 인간 배아 줄기 세포로부터 인간 심장 엽 성의 전구 물질과 Cardiomyocytes의 효율 유도

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

포유 동물 세포의 세포주기의 위치 분석

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

포유류의 링커 - 히스톤 Subtypes의 표현 분석

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

포유류의 세포에서 Endoplasmic 소포체 현지화 mRNAs의 시각화

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

배양 된 포유 동물 세포의 생물 학적 경로의 유비퀴틴과 유비​​퀴틴 같은 시스템 레귤레이터를 확인하는 siRNA를 상영

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

효율적인 포유 동물 세포 발현 및 결정화에 대한 세포 외 당 단백질의 단일 단계 정화

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