Transcript File

Unit III Lecture 3
B. Tech. (Biotechnology) III Year
V th Semester
EBT-501, Genetic Engineering
Unit III
• Gene library: Construction cDNA library and genomic
library, Screening of gene libraries – screening by DNA
hybridization, immunological assay and protein activity
• Marker genes: Selectable markers and Screenable
markers, nonantibiotic markers,
• Gene expression in prokaryotes: Tissue specific
promoter, wound inducible promoters, Strong and
regulatable promoters; Increasing protein
production;
• Fusion proteins; Translation expression vectors; DNA
integration into bacterial genome; Increasing secretions;
Metabolic load,
• Recombinant protein production in yeast:
Saccharomyces cerevisiae expression systems
• Mammalian cell expression vectors: Selectable markers;
One of the aim of rDNA technology
Synthesis of large quantities of protein,
either to
study its properties or
because it has commercial value
In such instances, detectable synthesis is
not sufficient: rather, it must be
maximized.
Factors affecting the expression of
cloned genes.
Promoter strength
Transcriptional termination
Plasmid copy number
Plasmid stability
Host-cell physiology
Translational initiation sequences
Codon choice
mRNA structure
Maximizing protein synthesis by use of
phage promoters in E.coli
There are three reasons for using a phage promoter.
First, such promoters are very strong, enabling large
amounts of RNA to be made in vitro.
Secondly, the phage promoter is not recognized by the E.
coli RNA polymerase and so no transcription will occur
inside the cell. This minimizes any selection of variant
inserts.
Thirdly, the RNA polymerases encoded by phages such as
SP6, T7 and T3 are much simpler molecules to handle
than the E. coli enzyme, since the active enzyme is a
single polypeptide.
Control of expression of chloramphenicol
acetyltransferase (CAT) in E. coli by three
different promoters.
The levels of CAT are expressed as μg/mg
total protein.
Vectors with strong, controllable promoters
are used to maximize synthesis of cloned
gene products
• much of the interest in the application of
recombinant DNA technology lies in the
possibility of facile synthesis of large quantities
of protein, either to study its properties or
because it has commercial value.
• Many gene products can be toxic to the host cell
even when synthesized in small amounts.
Strategy for regulating the expression of genes
cloned into a pET vector
Tissue specific promoters
Several types of promoters regulate gene
expression eg.
Constitutive promoters
Tissue-specific or development-stagespecific promoters
Inducible promoters
Synthetic promoters
Tissue specific promoters
Are promoter sequences on DNA of eukaryotic plant and
animal cells, which enable the expression of
particular gene in the specific cell type
As cells of an organism contain same genetic
information, some genes are turned on and others are
turned off at different locations and times during the life
cycle of an organism.
The transgenes driven by these type of promoters will
only be expressed in tissues where the transgene
product is desired, leaving the rest of the tissues in the
plant unmodified by transgene expression
Some examples of tissue specific promoters in plants
are
beta-amylase gene or barley hordein gene promoters (for seed
gene expression)
tomato pz7 and pz130 gene promoters (for ovary gene
expression)
tobacco RD2 gene promoter (for root gene expression)
banana TRX promoter
Promoters used in animal cells
Promoters and enhancer sequences are
used for driving transgene in different
animal
Some examples of tissue specific
promoters in animals are
cytomegalovirus immediate-early gene
promoter (CMV)
human desmin (Des)
human alpha-myosin heavy chain (α-MHC)
rat myosin light chain 2 (MLC-2)
human cardiac troponin C (cTnC)
Gene transfer to animal cells
Why animal cells are used for cloning
recombinant proteins
because they perform authentic posttranslational modifications not carried out
by bacterial cells and fungi
Objectives of gene transfer in animal cells
Study promoter function, reporter gene expression
regulation
Expression of recombinant proteins in cultured cell line
Improve the quality of farm animals e.g. improvement of
yield and quality of milk, meat, wool etc.
Express large quantities of foreign proteins in milk, serum or
blood of animals. Animals are referred as bioreactors and
the approach is called molecular farming or gene farming
To correct the function of nonfunctional genes (causing
genetic disorders) by introducing normal or functional
copies of genes, referred as gene therapy
Creation of specific cell lines or transgenic animal deleted
with known gene to study its importance in development
process. Such animals are called Knock out
Transfection Methods
Calcium phosphate precipitation
Dimethylaminoethyl-dextran) DEAE-dextran
mediated transfection
Uptake DNA by endocytosis of complex of DNA and
polycationic
Lipofection
Using liposomes
Fusion with bacterial protoplast with cultured cells
with PEG
Electroporation
Microinjection
Viral vectors
Vector systems used for transfection
Viral vectors
Papova virus
SV40 virus
SV40 transducing vectors
Late replacement vectors
Early replacement vectors
SV40 plasmid vectors
Non replicating vectors (passive transfecting vectors)
Retro Viruses
Vaccinia viruses
Adeno Viruses
Baculo viruses (For Transfecting insect cell line) or P
Element vectors
Mammalian Artificial chromosome
Baculo viruses (for expression in insect cell line)
Mammalian Cell lines expression
systems
•Two modes of expression - transient and stable.
•Cell lines used. Three cell types are dominant in transient expression: human
embryonic kidney (HEK), COS and baby hamster kidney (BHK), whilst CHO
(Chinese hamster ovary) cells are used predominantly for stable expression.
•Mammalian expression vectors. Eukaryotic origin of replication is from an
animal virus: e.g. Simian virus 40 (SV40). Popular markers for selection are
the bacterial gene Neor (encodes neomycin phosphotransferase), which confers
resistance to G418 (Geneticin), and the gene, encoding dihydropholate
reductase (DHFR). When DHFR is used, the recipient cells must have a
defective DHFR gene, which makes them unable to grow in the presence of
methotrexate (MTX), unlike transfected cells with a functional DHFR gene.
Promoter sequences that drive expression of both marker and cloned
heterologous gene, and the transcription termination (polyadenilation signals)
are usually from animal viruses (human CMV, SV40, herpes simplex virus) or
mammalian genes (bovine growth hormone, thymidine kinase).
•Strategies for co-expression of two cloned genes.
Expression Vectors for Mammalian
Cells
Sometimes required for difficult-toexpress
proteins or for “complete
authenticity” (matching glycosylation and
sequence)
• Cells are typically derived from the
Chinese Hamster Ovary (CHO) cell line
• Vectors usually use SV-40 virus, CMV or
vaccinia virus promoters and DHFR
(dihydrofolate reductase) as the selectable
marker gene
Mammalian Expression
• Gene initially cloned and plasmid propagated in bacterial cells
• Mammalian cells transformed by electroporation (with linear
plasmid) and gene integrates (1 or more times) into random
locations within different CHO chromosomes
• Multiple rounds of growth and selection using methotrexate to
select for those cells withhighest expression & integration of DHFR
and the gene of interest
Expression System Selection
• Choice depends on size and character of
protein
– Large proteins (>100 kD)? Choose eukaryote
– Small proteins (<30 kD)? Choose prokaryote
– Glycosylation essential? Choose baculovirus
or mammalian cell culture
– High yields, low cost? Choose E. coli
– Post-translational modifications essential?
Choose yeast, baculovirus or other eukaryote
Mammalian Systems
Disadvantages
Advantages
Selection takes time
Can express large
(weeks for set-up)
proteins (>50 kD)
Cell culture is only
Correct glycosylation
sustainable for limited
& signal peptide
period of time
removal, generates
Set-up is very time
authentic proteins
consuming, costly,
modest yields
Has chaperonins to
help fold “tough” prtns
pcDNA1.1
The vector pcDNA1.1/Amp contains the
SV40 and polyoma origins, a transcription
unit comprising the human
cytomegalovirus promoter and SV40
intron/ polyadenylaton site, an interstitial
polylinker to insert the transgene and the
ampicillin-resistance marker for selection
in E. coli
Saccharomyces cerevisiae as a MODEL SYSTEM
• 1997 – first eukaryotic organism sequenced
• 6607 ORF’s (see below)
• Saccharomyces Genome Database
As of Aug 12, 2009
4821 ORFs, 72.97%
975 ORFs, 14.76%
811 ORFs, 12.27%
Yeast systems for heterologous
expression: Saccharomyces cerevisiae
Eukaryote, unicellular, GRAS (Generally Regarded As Safe), capable of
performing post-translational modifications. Excellent recombinant technology:
vectors, markers, methods for transformation and gene manipulation, homologous
recombination of cloned sequences by single cross over (insertion) and double
cross over
Intracellular expression - higher protein yields, but more difficult extraction and
purification. Additional potential problems with:
a/ co- and post-translational processing of proteins at N- and C-termini.
b/ proteolytic degradation
c/ addition of tags might result in aggregation and insolubility
Secretion
The yeast secretory pathway is very similar to that in higher
eukaryotes.
N-terminal signal sequences for co-translational translocation of
screted proteins into the ER are removed by a signal peptidase.
Examples of popular signal sequences used for secretion of
heterologous proteins -these of Pho5, Suc2 and the a -factor.
Modification by N-linked (to asparagine) and O-linked (to serine/threonine)
glycosylation. Hyperglycosylation (outer chain extension) in the yeast
Golgi is not typical of mammalian cells. Yeast proteins only modified by
mannosylation (no other sugars).
Continue….
Specific problems with secretion of heterologous proteins
Hyperglycosylation can inhibit reactivity with AB, or render proteins
immunogenic (a problem for the production of therapeutic glycoproteins).
The obvious solutions: glycosylation mutants (mnn1, mnn9) or elimination of
potential sites for glycosylation. Alternatively use other yeast species like P.
pastoris.
The cell wall permeability can be a limiting factor. Some cell wall mutants have
higher cell wall porosity and release, as a result, heterologous proteins
better.
Folding of secreted proteins in the ER and involves accessory proteins such as
BiP (the product of KAR2), and PDI (protein disulphide isomerase).
Overexpression of these genes has been beneficial in some cases.
Proteolytic processing could be limited by insufficient amounts of required
processing enzymes, and in particular the products of SEC11, KEX2,
STE13 and KEX1 in cases of multicopy expression of proteins. Again might
need to overexpress some of these genes.
Baculovirus
• Baculovirus are present in invertebrates primarily insect
species
• They are not infectious for vertebrates & plants
• Genome is covalently closed circular double stranded of 134
kbp, due to its small it can accommodate large fragments of
foreign DNA
• They are divided into two groups on the basis of their structure
as-:
Nucleopolyhedroviruses (NPV)
Granuloviruses
These NPV are mainly used as expression vectors i.e.
Autographa californica NPV (AcMNPV) isolated from the
larva of the alfalfa looper
Contd..
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Baculovirus expression system based upon the
ability to propagate AcMNPV in insect cells
Uses many of the protein modification, processing
and transport systems present in higher eukaryotic
cells.
Virus that can be propagated to high titers adapted
for growth in suspension cultures
obtain large amounts of recombinant protein with
relative ease
Baculovirus are noninfectious to vertebrates and
their promoters are inactive in mammalian cells.
Baculovirus expression
system
• Recombinant baculovirus have become widely used
as vectors to express heterologous genes in cultured
insect cells and insects larvae
• Heterologous genes placed under the transcriptional
control of the strong polyhedrin promoter of the
Autographa californica polyhedrosis virus (AcNPV)
• Based on site specific transposition of an expression
cassette (pfast Bac with gene of interest) into a
baculovirus shuttle vector (bacmid)
Steps in recombinant
baculovirus production
• Clone the gene of interest in pfast Bac donor plasmid
• Expression cassette in pfast Bac is flanked by left and right
arms of Tn7 and also an SV40 polyadenylation signal to form a
miniTn7
• Cloned pfast Bac is transformed in E.coli host strain (DH10Bac)
which contains a baculovirus shuttle vector bacmid having a
mini-attTn7 target site
• Helper plasmid which allows to transpose the gene of interest
from pfast to bacmid (shuttle vector)
• Transposition occurs between the mini-att Tn7 target site to
generate a recombinant bacmid
• This recombinant bacmid can now be used to transfect insect
cell lines.
Baculovirus Expression
Baculovirus Systems
Advantages
Disadvantages
Grow very slowly (1012 days for set-up)
Cell culture is only
sustainable for 4-5
days
Set-up is time
consuming, not as
simple as yeast
Can express large
proteins (>50 kD)
Correct glycosylation
& signal peptide
removal
Has chaperonins to
help fold “tough” prtns
Very high yields, cheap