Experession vectores ppt bijan zare

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Information about Experession vectores ppt bijan zare
Health & Medicine

Published on March 2, 2014

Author: bijanzare

Source: slideshare.net

Description

Vectors that used to cloning and expressing of a gene in a suite host.

Expression Vectors Biotechnology Department Pharmacy college Tehran university of medical science Bijan zare November-2010 -15 1

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H1 Design of Plasmid Vectors T7 promoter RBS Start codon MCS Ampr Transcription terminator T7 expression vector ori 6

Strong and tunable promotor (high (affinity to RNA polimeras (lac (lacUV5. 1 trp.2 (tac (-10 lac + -35 trp.3 (p¹(left promotor of λ. 4 ( T7 promotor ( used in pET system. 5 7

Five promoters frequently used in expression vectors. The lac and trp promoters are shown upstream of the genes that they .normally control in E. coli 8

.Strong and weak promoters 9

Inducible Expression Vectors Protein produced in a large quantity in bacteria can be toxic, so it is advantageous to keep a cloned gene repressed .before expressing it Solution: keep the cloned gene turned off by placing it downstream of an inducible promoter that can be turned . off IPTG strongly induce lac promoter 10

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Example of inducable vector pCP3 is an inducible vectore In 28°c CI inhibite P¹ and plasmid is low copy number ( In 42°c increase in copy number(≈12 fold of total protein In pCP3 was T4 DNA ligase 20% 12

Phage λ A phage λ virion has a head, which contains the viral DNA genome, and a tail, which functions in infecting E.coli .host cells Advantages over plasmids: They infects cells much more efficiently than plasmids transform cells. The yield of .clones with vectors usually higher Because of its efficiency, phage λ is often .used in library construction Viral Genome 13

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Expression ?Why You want the cloned gene to make its product, normally a . protein .Identifying gene from library requires expression .To overproduce the protein and purify it . For in vivo studies of the protein 23

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The use of an expression vector to achieve expression of a foreign .gene in E. coli 25

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‫اطلاعات مفید در طراحی حامل بیان‬ ‫پایداری ذاتی پروتیین هدف‬ ‫ وجود پیوندهای دی سولفیدی‬‫ حضورآمینواسیدهای مختلف در ‪N‬ترمینال‬‫ اعدم حضور توالی ‪ PEST‬در توالی پروتیین‬‫ راه کار اتصال پروتیین هدف به یک پروتیین با ثبات میزبان‬‫)‪(fusion protein‬‬ ‫ استفاده از میزبان فاقد پروتئاز)البته کار ساده ای نیست بدلیل‬‫اعدم شناخت کافی از ژنتیک تمام پروتئازها – نقش خانه تکانی‬ ‫آنزیم ها(‬ ‫92‬

‫جایگاه نهایی پروتیین هدف‬ ‫ در پایداری پروتیین اثر دارد‬‫ در افزایش بیش از معمول بیان پروتیین‬‫- در تخلیص پروتیین نقش دارد‬ ‫03‬

‫بار متابولیکی حامل بر میزبان‬ ‫کپی شمار حامل‬ ‫راندمان ترجمه در ارگانیسم میزبان‬ ‫‪codon usage‬‬ ‫میزان مصرف اکسیژن‬ ‫ترافیک در مسیر صدور پروتیین های ترشحی‬ ‫نهایتا کاهش در رشد سلول‬ ‫13‬

Fusion Proteins Stability Identification Purification Quantification 32

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pLysS pLysE host strains they provides a small amount of T7 lysozyme, a natural inhibitor of T7 RNA polymerase . The presence of either pLysS or pLysE increases the tolerance of λDE3 lysogens for plasmids with toxic inserts 39

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Three of the problems that could be encountered when foreign genes are expressed (in E. coli: (a introns are not removed in E. ;coli b) premature) termination of transcription; (c) a problem with .codon bias 64

‫حامل ها و میزبانهای یوکاریوتی‬ ‫‪‬‬ ‫‪‬‬ ‫‪‬‬ ‫‪‬‬ ‫‪‬‬ ‫‪‬‬ ‫‪‬‬ ‫‪‬‬ ‫‪‬‬ ‫56‬ ‫مخمر‬ ‫یوکاریوت تک سلولی‬ ‫ژنتیک و بیوشیمی شناخته شده‬ ‫قابل رشد در اشل آزمایشگاهی و صنعتی‬ ‫پروموتور قوی ومتعدد‬ ‫وجود پلسمید 2 میکرومتری‬ ‫انجام پردازش بعد از ترجمه‬ ‫امکان ترشح پروتیین و سهولت تخلیص پروتیین‬ ‫مصرف در صنایع غذایی)بی خطر)‬

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Comparison between a typical glycosylation structure found on an animal protein and the structures synthesized by P pas/oris and S. .cerevisiae 71

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H4 Eukaryotic Vectors H4-3 Baculovirus Infects insect cells The strong promoter expressing polyhedrin protein can be used to overexpress foreign genes engineered. Thus, large quantities of proteins can be . produced in infected insect cells Insect expression system is an important .eukaryotic expression system

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H4 Eukaryotic Vectors H4-4 Mammalian viral vectors SV40: 5.2 kb, can pack DNA fragment . similar to phage l : Retroviruss single-stranded RNA genome, which copy . to dsDNA after infection Have some strong promoters for gene expression Gene therapy 79

.Structure of basic MMLV-based retroviral vectors The positions of the LTRs, internal promoter sequence (Pro), reverse orientation promoter (orP) polyadenylation site (AA), and coding regions of the therapeutic gene (solid areas) are indicated 80

Strategy for generating recombinant adenoviral vectors. The therapeutic gene is inserted into a cytomegalovirus (CMV) expression cassette multiple-cloning site (MCS) in the adenovirus (Ad) shuttle plasmid (pAdCMVLink), which contains the left-hand portion of the adenovirus genome minus the E1 gene (0–1 mu and 9–16 mu). The plasmid is transfected with the adenovirus genome minus 59 inverted terminal repeat (ITR) region into 293 cells, where the recombinant virus can .propagate 81

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