1 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 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

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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.