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Vectors that used to cloning and expressing of a gene in a suite host.
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Expression VectorsExpression Vectors
Biotechnology DepartmentBiotechnology Department
Pharmacy collegePharmacy college
Tehran university of medical scienceTehran university of medical science
Bijan zareBijan zare
1515- - November-2010November-2010
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T7 promoter
RBS
Start codon
MCS
Transcription terminator
Ampr
ori
T7 expression
vector
H1 Design of Plasmid Vectors
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Strong and tunable promotor(high affinity to RNA polimeras)
1 .lac (lacUV5)2.trp
3.tac (-10 lac + -35 trp)4 .p¹(left promotor of λ)
5 .T7 promotor ( used in pET system)
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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.
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Strong and weak promoters.
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Inducible Expression Vectors
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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
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Example of inducable vector
pCP3 is an inducible vectoreIn 28°c CI inhibite P¹ and plasmid is low copy number In 42°c increase in copy number(≈12 fold)
20% of total protein In pCP3 was T4 DNA ligase
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Phage λ
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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
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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 .
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The use of an expression vector to achieve expression of a foreign gene in E. coli.
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اطالعات مفید در طراحی حامل بیان
پایداری ذاتی پروتیین هدفوجود پیوندهای دی سولفیدی-
ترمینالN- حضورآمینواسیدهای مختلف در در توالی پروتیینPEST- عدم حضور توالی
- راه کار اتصال پروتیین هدف به یک پروتیین با ثبات میزبان (fusion protein)
استفاده از میزبان فاقد پروتئاز)البته کار ساده ای نیست -بدلیل عدم شناخت کافی از ژنتیک تمام پروتئازها – نقش
خانه تکانی آنزیم ها(
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جایگاه نهایی پروتیین هدف
- در پایداری پروتیین اثر دارد- در افزایش بیش از معمول بیان پروتیین
- در تخلیص پروتیین نقش دارد
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بار متابولیکی حامل بر میزبان
کپی شمار حاملراندمان ترجمه در ارگانیسم میزبان
codon usage میزان مصرف اکسیژن
ترافیک در مسیر صدور پروتیین های ترشحی نهایتا کاهش در رشد سلول
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Fusion Proteins
StabilityIdentificationPurificationQuantification
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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
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Three of theproblems that could beencountered when foreign genesare expressed in E. coli: (a)introns are not removed in E. coli;
(b )premature termination oftranscription; (c) a problem withcodon bias.
حامل ها و میزبانهای یوکاریوتیمخمریوکاریوت تک سلولی ژنتیک و بیوشیمی شناخته شدهقابل رشد در اشل آزمایشگاهی و صنعتیپروموتور قوی ومتعدد میکرومتری2وجود پالسمید انجام پردازش بعد از ترجمهامکان 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.
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H4-3 BaculovirusInfects insect cellsThe strong promoter expressing polyhedrin protein can be used to over-express foreign genes engineered. Thus, large quantities of proteins can be produced in
infected insect cells .Insect expression system is an important eukaryotic expression system.
H4 Eukaryotic Vectors
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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 expressionGene therapy
H4 Eukaryotic Vectors
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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
Strategy for generating recombinant adenoviralvectors. The therapeutic gene is inserted into a cytomegalovirus(CMV) expression cassette multiple-cloning site (MCS) in theadenovirus (Ad) shuttle plasmid (pAdCMVLink), which containsthe left-hand portion of the adenovirus genome minus theE1 gene (0–1 mu and 9–16 mu). The plasmid is transfectedwith the adenovirus genome minus 59 inverted terminal repeat(ITR) region into 293 cells, where the recombinant virus canpropagate.
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