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

1. Expression Vectors
Biotechnology Department
Pharmacy college
Tehran university of medical science
Bijan zare
November-2010 -15
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6. H1 Design of Plasmid Vectors
T7 promoter
RBS
Start codon
MCS
Ampr
Transcription
terminator
T7
expression
vector
ori
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7. 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
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8. 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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9. .Strong and weak promoters
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10. 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
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12. 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%
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13. 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
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23. 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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25. The use of an expression
vector to achieve
expression of a foreign
.gene in E. coli
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29. ‫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(‬
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30. ‫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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31. ‫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
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32. Fusion Proteins
Stability
Identification
Purification
Quantification
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39. 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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64. 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
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65. ‫حامل ها و میزبانهای یوکاریوتی‬
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‫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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71. 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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76. 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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79. 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
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80. .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
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81. 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
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