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Expression of cloned genes in cultured cells
Isik G. Yulug
Based on info in book:
Applied Molecular Genetics, R.L.Miesfeld,
Chapter 7, p. 175
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Expression of Recombinant Proteins:Vectors, promoters and reporters
Promoter MCS
VECTORSVectors for protein expression contain- the basic DNA elements for handling in E.coli - an eukaryotic promoter upstream of MCS - appropriate DNA elements for maintenance of the vector in target host cells (mammalian, yeast, plant, Drosophila)
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Cloning Vectors:Basic Components
Plasmids: naturally occurring bacterial ‘minichromosomes’
Vectors: Engineered derivatives of plasmids used forcloning
Origin of replication (Ori)– ColE1, p15a plasmids (copy number 1 - >50)– f1- filamentous phage– Eukaryotic- SV40
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Mechanism of plasmid selectionAntibiotics:Ampiciline
ChloramphenicolKanamycinTetracycline
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Cloning Vectors: Basic Components
Multiple cloning site (MCS)– Selection of unique restriction enzyme sites
Mechanism of insert detection– LacZ peptide (blue / white colour)– Antibiotic inactivation– (Luciferase, CAT)
Other features– T7 / SP6 promoters– Transcription of insert (? bidirectional)– Sequencing primer binding sites (M13, T7)
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Cloning Vectors: Specialized features
Specialized vectors exist to perform different functions
ExpressionExpression cassettes - protein expression;Require promoter, initiation & termination codons, RBS(Kozak,polyA tail [3’UTR])Fusion proteins - purification systems - His tag, GST, Maltose
Reporter genesanalysing promoters, enhancers, translational processes
SequencingProduction of ssDNA
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Rare cellular proteins can be made in large amounts using cloned DNA
Previously, could purify 0.1 g of an abundant protein if started with several hundred grams of cells – then could analyze it structurally, biochemically, use it to raise antibodies, etc.
But impossible to do this previously for all the proteins present in small amounts
Now can do this via DNA cloning and engineering
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To make large amounts of protein in vitro, clone the coding sequence for a protein into an expression vector:
Vectors must have what?? so the gene is transcribed efficiently in the bacterial cell to make mRNAs
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To make large amounts of protein in vitro, clone the coding sequence for a protein into an expression vector:
Vectors must have promoters and gene regulatory
sequences so the gene is transcribed efficiently in the
bacterial cell to make mRNAs
The mRNA produced must have all translational control
signals – what?
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To make large amounts of protein in vitro, clone the coding sequence for a protein into an expression vector:
Vectors must have promoters and gene regulatory sequences so the gene is transcribed efficiently in the bacterial cell to make mRNAs
The mRNA produced must have all translational control signals – what?
Shine-Dalgarno sequence, AUG, stop codons
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PromoterTranscription of your DNA into RNA
Cells with RNA Polymerase e.g. T7 RNA polymerase/pET)
Ribosome binding site (RBS)For Translation of your RNA into protein
Appropriate cell Host
Antibiotic selection/Medium (Amp, Kan, Tet) - LB, 2xYT
Storage/transformation of clones
Induction – turns on protein expressionTemp (37°, 25°), IPTG (0.1 - 1.0mM)
Detection of your target protein – assay?
From Gene to Functional protein: Expression checklist
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10_32_expressionvector.jpg
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Different expression vectors designed to work in different cells – yeast, insect, mammalian – with the appropriate regulatory sequences.
Cells then produce large amounts of protein, which can be purified for study.
Also, large amounts of proteins important in medicine can be produced – pure and free of contamination:
e.g. factor VIII protein, insulin, viral coat proteins, for vaccine production, etc.
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10_33_gene_protein.jpg
Can move from gene to protein, or protein to gene
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Promoters (Table 7.1. Fig. 7.1)
• Ubiquitous promoters (viral origin) - These promoters work well in all cell types
• Minimal promoters - low level of expressionExample: TK promoter from HSV
• Strong promoters - high level of expression Example: CMV- very strong
SV40 - moderate
Basic elementsTransactivator binding sitesMCS
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Cell type specific promoters:These are derived from genes, which are specifically expressed in certain tissues
- expression in liver, saliva, skin, etc.- used in transgenic organisms to restrict
expression of the trans-gene in certain
tissues
Example: myosin promoter in muscles
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• Inducible promoters –used broadly in functional analyses
• Promoters induced by endogenous factors :- hsp70 – by elevated temperature (in Drosophila)- MMLV- by steroid hormones (in mammalian cells)- GAL1-10 (UAS) – by galactose in yeast
• Synthetic inducible promoters - derived from unrelated organisms - regulated by a simple mechanism- can be very precisely controlled
Example:
-Tetracycline – antibiotic inducible system -Ecdysone - insect hormone inducible system
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The tetracycline system
- TET mutant can not bind DNA in the presence of tetracycline. Upon removal of tetracycline the TET domain binds DNA and activates promoters
- Reporter plasmids are designed to respond to binding of TET
- Tet-off works well in tissue culture cells, not that well in whole organisms because of side effects and degradation of tetracycline
Tet-off (Fig.7.6 a)
- Tetracycline receptor (TET) is a bacterial DNA binding protein
- The TET DNA binding domain is fused to a transactivation domain (TA)
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MCS
CMV promoter
TET
TET TA
TA
TETTA
Plus tetracyclin
MCSTET
TATET
TA
No tetracyclin
The tetracycline system consists of two plasmids. One expresses the TET-TA fusion protein. The other one contains the protein encoding sequence under the control of TET-TA responsive promoter
TET-OFF
(TetRE)7
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Tet-on (Fig.7.6 b)-This is an alternative TET system, which works well in both tissue culture cells and in whole organisms
-TET wt can not bind DNA in the absence of tetracycline. Upon addition of tetracyline the TET domain binds DNA and activates promoters
!! Both tet-off and tet-on systems can be leaky in certain cell types
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CMV promoterTET TA
MCS
TETTA
TETTA
No tetracyclin
TET-ON
MCS
Plus tetracyclin
TETTA
TETTA
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The ecdysone system
Ecdysone is a steroid hormone, which works in insects only
-One plasmid is expressing the Ecdysone receptor
-The reporter plasmid contains the protein encoding sequence
under the control of Ecdysone receptor.
-This is a very tightly regulated system
-Response to ecdyson is quantitative
-Virtually no side effects
-Ecdysone is metabolized similarly to other steroid
hormones
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MCS
Expression plasmid
Ecdysone receptorCMV promoter
EcR EcREcdysone
EcR EcRMCS
Expression plasmid
EcR EcR
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Engineered genes can reveal when and where a gene is expressed – many genes about which nothing is known
Easiest to use a reporter gene to study an unknown protein’s expression pattern
Experimentally join the controlling sequences of protein X to a gene that makes a product easy to follow in the cell (e.g. b-galactosidase)
The level, timing, and cell specificity of the reporter gene product will tell you about protein X
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REPORTER GENES
The primary objective is to identify gene regulatory sequences by testing a large number of deletion and point mutants in an in vivo cell transfection assays.Promoter mapping studies can be done by creating artificial “reporter gene” by fusing regulatory region of the test gene to a heterologous gene coding sequences that direct the synthesis of a readily detectable protein product.
The steady state level of the reporter protein in the transfected cell is directly correlated to the steady state level of reporter mRNA, then it is possible to use protein based reporter assays for promoter mapping experiments.
Reporter genes;1. must encode a protein activity that is similar to one already present in the cell.2. The protein assay should be sensitive enough, reproducible and easy to perform.
3. The reporter protein function should not interfere with host cellular processes in a way that will alter intracellular signaling pathways or metabolic rates.
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Reporter gene constructs:
• When trying to determine what are the cis-acting regulatory regions that control the expression (transcription) levels of a gene, is usually routine to clone a large piece of DNA containing these sequences and hook it to a downstream sequence that encodes an easy-to-measure protein called a reporter.
• This is a lot more convenient and accurate than trying to estimate the
absolute levels of mRNA.
• In vivo transgene reporter expression driven by the promoter of
interest will reveal the temporal/spatial pattern of expression of the
gene, measured by an easier method than the alternative in situ
hybridization.
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Reporter gene constructs:
The most used reporters are:• bacterial -galactosidase,• chloramphenicol acetyl transferase (CAT),• fire-fly luciferase• and most recently, the green fluorescent protein (GFP) which
allows measurements in living cells or organisms.
Part of the whole of a protein rather than a promoter can also be fused in frame to this reporters to find out where it localizes. Reporter gene must not being expressed endogenously in the cell types that you are working on, so their background should be null.
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green fluorescent protein = GFP (the protein that gives luminescent jellyfish their greenish glow…
For GFP, can often attach it to the end of the protein of interest (creating a fusion protein), then watch for the greenish glow to see where protein X is….
Reporter Genes (GFP)
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In vivo detection of protein expression: Green Fluorescence Protein- Proteins are expressed as GFP-fusions- GFP is fluorescent- Excellent assay for non-disruptive confirmation of expression,
protein localization, etc.- There are several GFP mutants which emit different color light - Vectors are designed for easy cloning of proteins as GFP-fusions
Reporter Genes (GFP)
GFP MCSCMV promoter
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transfect
Example: Localization/morphology studies
Protein A is fused to green GFP
Protein B is fused to red GFP
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10_35_GFP.jpg GFP used to show living neurons in a live fruit fly embryo – attached to a promoter active only in neurons
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Green Transgenic MiceOsaka University spermatologist Masaru Okabe and colleagues at Osaka's Research Institute for Microbial Diseases added a form of a gene for green fluorescent protein (GFP) from the jellyfish Aequorea victoria into mice, producing animals that are green through and through when exposed to blue light. "It's very beautiful," Okabe says. The gene had previously been transferred into fruit flies and zebrafish, but not mammals.
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Bacterial Expression of GFP Variants
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Disadvantages of GFP- not as sensitive and quantitative as CAT, luciferase or -GAL
- Can interfere with function of the protein (GFP) may block an important active site at the end of the polypeptide)
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Transfer of DNA to mammalian cells: Transfection
How to put DNA (or antisense agents) in eukaryotic cells
• Physical (stealth) transfection – very easy and popular, Good for transfecting culture cells- Ca3(PO4)2 precipitation
= Can activate signal transduction pathways or stress responses- DEAE dextran precipitation
= alternative of Ca3(PO4)2 precipitation - Liposomes
= the most efficient and gentle method= expensive= high hopes for drug delivery (incl. anti-sense drugs) in
vivo. There are attempts to make cell type specific liposomes
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Liposome-mediated transfection: A mixture of a polycationic lipid and a neutral lipid will result in the formation of unilamellar liposome vesicles that have a net positive charge due to the highly-positive amine head groups on these molecules. Liposomes have become the method of choice for carrier molecules in routine cell line DNA transfections because liposome-mediated gene delivery is technically easy, highly reproducible, and very efficient.
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Transfer of DNA to mammalian cells: Transfection
• Electroporation= uniform high efficiency with most cell types= very easy and gentle
• “The Gene gun”- DNA is shot at very high speed into to cells- It is used on whole organisms- Attempts to apply it in gene therapy- Routinely used for transformation in plants
• Injection- 100 % efficiency, very laborious- Most frequently used in making transgenic animals
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Electroporation
Electroporation is the most versatile method of DNA transfection because it has been shown to work for such a wide variety of cell types, which includes primary cells from tissue isolates, plant protoplasts and bacterial cells. By varying the electric field strength, and the length of time the cells are exposed to the electric field, it is possible to optimize electroporation parameters for essentially any cell type.
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Electroporation
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• Microinjection
• Gene gun
DNA can be inserted into a cell by:
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Microinjection
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In vitro development of rabbit embryos.The 8-cell (A), morula (B)blastocyst stage (C) embryos
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Types of transfection
Transient transfection- The vector is NOT incorporated in the genome- Vectors are lost at high rate in cells - The cloned protein is expressed temporarily- Easy, good for simple short term experiments in cell culture
Stable transfection- The vector is randomly incorporated in the genome- Stably transfected cells are selected by drug resistance technology- Good for long term experiments- Used in transgenic technologies- The promoters can be tightly regulated and proteins are expressed at will
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Transient Transfection Assays
For many types of gene expression assays, it is possible to transfect cell lines with an appropriate reporter gene, and then collect the data 24-48 hours later. Relative to the generation time of most cell lines (~16-24 hours), this represents a relatively short time period between DNA transfection and cell harvesting, and hence the term "transient" transfection.
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Transient transfection protocols need to be optimized for each cell type being analyzed due to inherent differences in DNA uptake efficiencies.In the example shown in the next slide, the electroporation conditions of 250V and 1180 mF would be chosen as optimal for efficient transient transfection of this cell type.
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Stable DNA Transfection
Stable DNA transfection of cell lines is basically done
the same way that transient transfections are, with the
exception that stable integration of the transfected DNA
needs to be positively selected for by including a marker
gene on the expression vector.
Most often the marker gene encodes an enzyme that
inhibits the function of a toxic compound. A description
of the most commonly used dominant selectable marker
genes for stable transfection of eukaryotic cell lines is
given in the next slide.
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Selection of positive stable transfectants. Selection markers are genes, which neutralize a toxic drug.
A list of frequently used selection markers in higher eukaryotes.
neoR – neomycin hygR – hygromycinpacR – puromycinzeoR – zeomycin
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Stable DNA transfection of cell lines is basically done the same way that transient transfections are, with the exception that stable integration of the transfected DNA needs to be positively selected for by including a marker gene on the expression vector. Most often the marker gene encodes an enzyme that inhibits the function of a toxic compound. A description of the most commonly used dominant selectable marker genes for stable transfection of eukaryotic cell lines is given below.
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Transfer of DNA to mammalian cells: Transfection
• Viral gene transfer.= most sophisticated high efficiency method= can be cell-type specific or general vector
• Retroviral gene transfer (The M13 like vectors for mammalian cells)
The retroviral genome:
LTRLTR gag pol env ψ
LTR promoter
Structural genes, pol is Reverse Transcriptase (RT)
Element, required for packaging of viral particles
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Retroviral vectors (See Fig. 7.4.)LTR and ψ elements are inserted in a plasmid
LTRLTR neoR ψCMV MCS
AbR ColE1
Packaging cell lines
These cells contain retroviral structural genes, no ψ (Element, required for packaging of viral particles ) or LTR element
Gag
Pol
env
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Production of retroviral particles (Fig. 7.4.)-Retroviral vectors are transfected in packaging cells-gag, pol, env will produce “retroviral” RNA using the LTR and
package it in “retroviral” particles. RT is incorporated in the particle
gagpol (RT)env
ψ
ψ
plasmid
ψ
ψ
ψ
ψ
Retroviral particles
Recombinant retrovirusproducing cell
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ψ
Retroviral particle
Retroviral particles infect target cells. RNA is converted to DNA by RT
ψ RNA
DNAψ
RT
Characteristics of retroviral transfection systems
- Very high efficiency- DNA is frequently integrated in the genomes - Major tool in research, rare attempts for gene therapy
Add media from producer cell line directly to media of host targets
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Expression and purification of recombinant proteins
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Protein Disease
a-antitrypsin EmphysemaEpidermal growth factor Wound healingInsulin DiabetesErythropoietin AnaemiaFactor VII HaemophiliaFactor IX HaemophiliaGrowth hormone Growth disordersTissue plasminogenactivator Heart attacks
Many human proteins are used to treat disease.
These are normally present in the body in trace amounts.
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Unique restriction sites
CATATG
Strongpromoter(regulatable)
StrongRBS(Ribosome
Binding Site)
Nde I site
e. g. Hind III
E. coli expression plasmids
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GGG ATGCCC TAC
TGA AAAACT TTT
NNNN CAT ATG
ACT TCGAA NNNN
PCR is used to create an Nde I restriction site at the start codon of the target gene
and another restriction site e. g. Hind III downstream of the stop codon.
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NNNCAT ATG TGA AGCTT NNN
The amplified form of the gene (the PCR product) has the restriction sites at each end.
Nde I
Hind III
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CATATG
Nde I Hind III
Inserting the amplified gene into the expression vectorplaces the start codon at the optimum distance from the RBS(2 - 11 bp upstream).
NNNCAT ATG TGA AGCTT NNN
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The amplified gene must be re-sequenced to ensure that there are no base changes.
Many heat-stable DNA polymerases have a high error rate.
Taq polymerase has no proof-reading exonuclease activity and frequently mis-incorporates bases.
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The pT7-7 system
T7 promoter
pT7-7 expression plasmid
Nde I HindIII
pGP1-2
Inducible T7 RNA polymerase
(KanR)
(AmpR)
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T7 RNA pol
- only recognizes the T7 promoter(a 23 bp sequence)
- initiates frequently
- transcribes DNA at 200bases/sec(4 x faster than E. coli RNA pol)Inducible T7
RNA polymerase
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Control
+ expression plasmid
Band of overproducedprotein.
E. coli cellsMW markers
29
45
205
kDa
Analysis of expression level by protein gel electrophoresis.
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Band of overproducedprotein.
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Affinity chromatography
The tag is retained by an affinity ligand, which is immobilized on chromatography beads.
tag protein X
Elute
Purified Fusion Protein
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Affinity chromatography
Expression vectors often encode protease cleavage sites between the tag and the cloned protein to enable removal of the tag after purification.
tag protein X
Protease Cleavage
Purified Protein X
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ATG CAC CAC CAC CAC CAT CAT ATGTAC GTG GTG GTG GTG GTA GTA TAC
Some expression vectors encode hexahistidine tags.
M H H H H H H M
RBS Start codon Nde I
Tagged proteins bind specifically to immobilised metal ion affinity columns.They can be purified in one step.
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MCS
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My expression system doesn’t work….
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Target protein can be produced at a high level[ up to 30% of total cellular protein]
Problems:
Overproduced proteins may form inclusion bodies,aggregates of incorrectly folded inactive protein.
Eukaryotic genes may have codon usagewhich is unfavourable for expression in E. coli.
Specialised host strains have plasmids with extra copiesof genes for rare tRNAs.
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Protein stability
Recombinant proteins do not accumulate if they are rapidly degraded by proteases. The N-terminal AA affects stability.
N-terminal AA Half-life
Met, Ser, Ala,Thr, Val, Gly 20 hours
Ile, Glu > 30 min
Tyr, Gln ~ 10 min
Pro ~ 7 min
Phe, Leu, Asp, Lys ~ 3 min
Arg ~ 2 min
Changing the first AA can improve stability.
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Frequently used tags in the E. coli system. Do not memorize all
Tag Description Affinity ligand His6- 6 histidines Ni++, Co++, Cu++, GST - glutathione-S-transferase glutathione PrA Protein A IgG
Mal Maltose binding protein Maltose CBP Calmodulin binding protein Calmodulin
E.coli expression systems:
Advantages-high levels of expression (1-10% of total cellular protein)-inducible-affordableDisadvantages-Proteins can lose characteristic activity (post-translational modifications)-Proteins can misfold and precipitate (chaperones are missing)
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Eukaryotic expression system
Baculovirus
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Alternatives of E. coli expression systems
Baculo-virus expression system
-Host cell line is Sf9 (insect), vector is baculovirus-Gene(s) of interest is cloned into recombinant baculo-viruses-Baculo-viruses infect insect host cells and express the proteins at very high levels
Advantages/Disadvantages of baculovirus:-most expressed proteins are biologically active -Can express large proteins, multi-subunit proteins -expensive and time-consuming
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• Baculovirus expression– 150 kb genome. – AcMNPV = Autographa califonica multiple
nuclear polyhedrosis virus– Insect cells.– Low frequency recombination inserts foreign gene
into viral genome.– Recombinant virus do not have coat protein.
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Advantages:• Baculovirus expressed proteins obtained from insect cell lysates are
often fully active and soluble,• The viral genome is large and can accommodate cDNA insertys of up
to 15 kb without compromising viral replication• Functional multi-subunit protein complexes can be assembled in vivo
and expressed at high levels by co-infecting cells with two or more
recombinant viral stocks• The virus has a very restricted host range and it is therefore safe to
handle and poses minimal environmental risk• Under optimal conditions: as much as 1-5 mg of protein can be
produced per liter of infected cells.
Disadvantage:
The initial isolation and characterization of the primary viral
recombinant can be chalenging.