Презентация на тему "Gene Expression Systems in Prokaryotes and Eukaryotes"

Презентация: Gene Expression Systems in Prokaryotes and Eukaryotes
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  • Формат
    pptx (powerpoint)
  • Количество слайдов
    96
  • Слова
    английский язык
  • Конспект
    Отсутствует

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  • Презентация: Gene Expression Systems in Prokaryotes and Eukaryotes
    Слайд 1

    Gene Expression Systems in Prokaryotes and Eukaryotes

    1 Expression studies Expression in Prokaryotes (Bacteria) Expression in Eukaryotes

  • Слайд 2

    2 Expression studies: 1. Analyzing Transcription - Northern blot - Micro array - real-time PCR - Primer extension 2. In vivo Expresion studies Use of report genes to study regulatory elements 3. Analyzing Translation - Western blot - immuno assays - 2D electrophoresis - proteomics

  • Слайд 3

    3 Studying Transcription Microarray technique – DNA chips

  • Слайд 4

    4

  • Слайд 5

    5 Studying Transcription Primer Extension

  • Слайд 6

    Promoter Studies

    6 Used reporter genes: Lac Z GFP Luciferase Promoter

  • Слайд 7

    7 Promoter studies by using reporter genes

  • Слайд 8

    Luciferase (luc) systems

    8 firefly species Photinus pyralis oxidation of compounds called luciferans ( ATP-dependent process) luciferans emit fluorescense Expressed luciferase catalyses mouse with a strain of salmonella Mice are injected with LUC+ salmonellas. Sensitive digital cameras allow non-invasive detection. For GT vectors pics look the same luminometer measurement

  • Слайд 9

    Green fluorescent protein (GFP)

    9 autofluorescent protein from Pacific Northwest jellyfish Aequorea victoria GFP is an extremely stable protein of 238 amino acids with unique post-translationally created and covalently-attached chromophore from oxidised residues 65-67, Ser-Tyr-Gly ultraviolet light causes GFP to autofluoresce In a bright green color Jellyfish do nothing with UV, The activate GFP by aequorin (Ca++ activated, biolumuniscent helper)

  • Слайд 10

    GFP expression is harmlessfor cells and animals

    10 GFP transgenic mice from Osaka University (Masaru Okabe) GFP construct could be used for construct tracking in living organism GFP labelled image of a human tumor. Vessel on the tumor surface are visible in black

  • Слайд 11

    Many more fluorescent proteins are engineered

    11 Engineered proteins are covering all the spectrum San Diego beach scene drawn with living bacteria expressing 8 different colors of fluorescent proteins.

  • Слайд 12

    Use of green fluorescent protein (GFP) as a reporter gene.

    12 Page 119

  • Слайд 13

    13 Analyzing Translation – Western Blot

  • Слайд 14

    2 D Electrophoresis

    14

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    Gene Expression

    15 Transcriptional start Translational start

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    16 Gene copy number: 1. Plasmid copy number: The copy-number of a plasmid in the cell is determined by regulating the initiation of plasmid replication. The initiation of plasmid replication may be controlled by: the amount of available primer (RNA) the amount of essential replication proteins the function of essential replication proteins. 2. Gene dosage -> number of genes integrated into chromosome - prokaryotic systems -> i.e. Transposons, phages, recombinantion - mainly eukaryotic systems

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    17 Incompatibility of plasmids: Not all plasmids are able to coexist in the same cell. Plasmids which have the same replication control functions are incompatible, and are assigned to the same incompatibility group (inc group). Plasmids of one incompatibility group are related to each other, but cannot survive together in the same bacterial cell, as only different kinds of plasmids are compatible. Ensures that we can make libraries -> just one plasmid taken up by one cell

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    Homologous integration into chromosome

    18 Insertion on Bacillus subtilis chromosome

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    Protein expression in prokaryotic systems

    19 www.qiagen.com So, this new story would be about vectors again. Bacterial expression vectors have some distinct features: Inducible promoter systems; Protein fusions including fused tags;

  • Слайд 20

    General advices for one who wants to produce gene expression in prokaryotes

    20 1. Do not forget to cut out the intron 2. Check orientation of insert 3. Do fusions with something In-frame Most obvious and common mistakes: 4. No Post-translation modification = no product activity

  • Слайд 21

    Introns

    21 www.wzw.tum.de/gene-quantification/ mrna.html Not an issue when you clone a cDNA

  • Слайд 22

    Orientation of insert (could go backward, if cloned with same-type sticky ends) – use incompatible sticky ends

    22 www.bch.bris.ac.uk/staff/ pfdg/ teaching/genes.htm

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    Fusion proteins.

    23 When expressing a fusion proteins, ensure that both of them are in the same reading frame www.bch.bris.ac.uk/staff/ pfdg/ teaching/genes.htm

  • Слайд 24

    PostTranslational modification

    24 Eukaryotic cells have Golgi system Prokaryotic cells do not have it nucleus Golgi

  • Слайд 25

    Efficiency of expression in E.coli

    25 Dependent of: 1. Type of transcription promoter and terminator 2. Affinity of mRNA and prokaryotic ribosome 3. Amount of copies of transgene and its localization (chromosome or plasmid) 4. Cellular localisation of the protein end-product 5. Efficiency of translation in the host organism 6. Stability of protein product in the host organism Systems could be optimized on gene to gene basis. No universal strategy possible

  • Слайд 26

    Factors affecting transcription

    26 Promoters (including regulated ones) PROKARYOTIC!!!! 2. Terminators PROKARYOTIC!!!!

  • Слайд 27

    Variations between prokaryotic promoters are minimal

    27 http://www.blc.arizona.edu/marty/ 411

  • Слайд 28

    Factors affecting translation

    28 1. Ribosome binding site (RBS) 2. Codon bias 3. Stability of the transcript

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    Ribosome binding site (RBS) =translation initiation site complimentary to 16S rRNA

    29

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    Codon Usage in E. coli & humans

    30

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    Codon Optimization Strategies

    31 Chemically synthesize new gene Alter sequence of the gene of interest to match donor codons to the codons most frequently used in host organism Express in different host choose host with better matching codon usage Use an engineered host cell that overexpresses low abundance tRNAs

  • Слайд 32

    Commercial E. coli strains encode for a number of the rare codon genes

    32 AGG/AGA (arginine), CGG (arginine), AUA (isoleucine) CUA (leucine)CCC (proline), and GGA (glycine) (AT-rich compatible) Rosetta or Rosetta (DE3) arginine (AGG, AGA) and proline (CCC) BL21 (DE3) CodonPlus-RP (GC-rich compatible) arginine (AGG, AGA), isoleucine (AUA) and leucine (CUA) BL21 (DE3) CodonPlus-RIL (AT-rich compatible)

  • Слайд 33

    Mitochondria and chloroplast genes

    33 Alterations in the Standard Genetic Code in Mitochondria Leu Thr Leu Leu Leu Leu CUU, CUC, CUA, CUG Ile Met Met Met Met Ile AUU Ile Met Ile Met Met Ile AUA Arg Arg Arg Ser Stop Arg AGA, AGG Stop Trp Trp Trp Trp Stop UGA Plants Yeasts Neurospora Drosophila Mammals Standard Code: Nuclear-Encoded Proteins CODON Mitochondria

  • Слайд 34

    Factors affecting protein stability

    34 Overall level of protease activity in bacterial cells 2. N-terminal amino acid affects protein half-life 3. Internal regions containing clusters of certain amino acids can increase proteolysis P prolineE glutamic acidS serineT threonine …. Mutate PEST aminoacids….

  • Слайд 35

    Protease-deficient host strains

    35 BL21, the work horse of E. coli expression, is deficient in two proteases encoded by the lon (cytoplasmic) and ompT (periplasmic) genes. It is dangerous to kill proteases, it makes E.coli grow much slowly as proteases needed for proper metabolism

  • Слайд 36

    Inducible bacterial promoters

    36 Why not to use constitutive, always strong promoter? Induction Because recombinant (alien) protein is often toxic for bacterial cell. Bacteria tend to expel harmful plasmids Bacterial grow takes time….

  • Слайд 37

    BL(DE3) inducible system and pET vectors (invented in 1984 by Bill Studier, on sale by Novagen)

    37 1) T7 RNA polymerase gene is integrated in chromosome under the control of a lac promoter and operator 2) lactose analogue, IPTG, causes the host to produce T7 RNA polymerase 3) The E. coli host genome also carries the lacI (repressor) gene pET23 Gene of interest is expressed from strong T7 promoter

  • Слайд 38

    Why repressor gene and gene of interest are expressed from different DNA molecules?

    38 Repressor gene expressed from chromosome; Gene of Interest expressed from plasmid If too high repressor no transcription (you need to increase expensive IPTG) If too low repressor promoter is leaky (active without IPTG) Repressor is in chromosome, because there it is best kept controlled there (no plasmid loss, not too high expression)

  • Слайд 39

    Where your expressed protein will be located?

    39 Inclusion bodies (insoluble) Cytoplasm (soluble) Periplasmatic space (soluble or insoluble) Secreted (!!) E.Coli can not do that

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    1. Inclusion bodies (most common case)

    40 -- Inclusion bodies are formed through the accumulation of folding intermediates rather than from the native or unfolded proteins. -- It is not possible to predict which proteins will be produced as inclusion bodies. -- Production of inclusion bodies not dependent on the origin of protein, the used promoters, the hydrophobicity of target proteins...

  • Слайд 41

    Electron micrograph of an inclusion body of the protein prochymosin in an E. coli cell

    41 Page 116 Protein Folding

  • Слайд 42

    Good side of inclusion bodies

    42 inclusion bodies can be accumulated in the cytoplasm to much higher level (greater than 25%) than production as soluble form; 2) inclusion bodies is initially isolated in a highly purified, solid, and concentrated state by simple physical operation (centrifugation). 3) inclusion bodies have no biological activity. For toxic proteins it may be the only one available; 4) inclusion bodies areresistant to proteolysis That results in the high yield of protein production.

  • Слайд 43

    SDS-PAGE analysis of recombinant protein produced as inclusion body

    43 hG-CSF mbel.kaist.ac.kr/research/ protein_en1.html

  • Слайд 44

    Recovery of proteins from inclusion bodies

    44 Is not a straightforward process, but road of trials and errors Solubilization Refolding Choice of solubilizing agents, e.g., urea, guanidine HCl, or detergents, plays a key role in solubilization efficiency -- Refolding is initiated by reducing concentration of denaturant used to solubilize IBs. Guandinium -- Refolding competes with other reactions, such as misfolding and aggregation (both are leading to bad results) -- Chaperones are helpful in refolding (including chemical chaperones)

  • Слайд 45

    Question of questions – how to purify your protein?

    45

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    Diversity of proteins could be exploited

    46 Columnchromatography Matrix particles usually packed in the column in the form of small beads. A protein purification strategy might employ in turn each of the three kinds of matrix described below, with a final protein purification Of up to 10,000-fold. Essential Cell Biology: An Introduction to the Molecular Biology of the Cell

  • Слайд 47

    Column chromatography

    47 Different proteins are retarded to different extents by their interaction with the matrix, they can be collected separately as they flow out from the bottom. According to the choice of matrix, proteins can be separated according to -- their charge, -- their hydrophobicity, -- their size, -- their ability to bind to particular chemical groups (!!) Essential Cell Biology: An Introduction to the Molecular Biology of the Cell

  • Слайд 48

    (A) ION-EXCHANGE CHROMATOGRAPHY

    48 Ion-exchange columns are packed with small beads that carry positive or negative charges retarding proteins of the opposite charge. The association between a protein and the matrix depends on the pH and ionic strength of the solution passing down the column. These can be varied in a controlled way to achieve an effective separation. Essential Cell Biology: An Introduction to the Molecular Biology of the Cell

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    (B) GEL-FILTRATION CHROMATOGRAPHY

    49 Gel-filtration columns separate proteins according to their size on tiny porous beads. Protein molecules that are small enough to enter the holes in the beads are delayed and travel more slowly through the column. Proteins that cannot enter the beads are washed out of the column first. Such columns also allow an estimate of protein size. Essential Cell Biology: An Introduction to the Molecular Biology of the Cell

  • Слайд 50

    (C) AFFINITY CHROMATOGRAPHY

    50 Affinity columns contain a matrix covalently coupled to a molecule that interacts specifically with the protein of interest (e.g., an antibody, or an enzyme substrate). Proteins that bind specifically to such a column can finally be released by a pH change or by concentrated salt solutions, and they emerge highly purified. Essential Cell Biology: An Introduction to the Molecular Biology of the Cell

  • Слайд 51

    Protein electrophoresis

    51 Essential Cell Biology: An Introduction to the Molecular Biology of the Cell

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    52 www.unizh.ch/.../Teaching_slide_shows/ Lambda/sld015.htm

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    Fusion proteins

    53 increase production level facilitate purification (taq) detection of expression (GFP fusion) Redirection of proteins (secretion -> signal peptidases) Surface display (for screening of libraries) Tandem arrays (for small peptides, toxic proteins,..)

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    Most widely used purification strategy – to produce your protein as a fusionwith something easily purifyable

    54 (Invitrogen, Life Technologies, Novagen, QIAGEN): 6xHIS Tag 1. This small addition rarely affects protein structure to a significant degree 2. Interaction so strong, it tolerates denaturing conditions (could be used for inclusion bodies purification)

  • Слайд 55

    Histidine: a charged aminoacid

    55 The affinity of this interaction is very high which allows protein purification to 95% in a single step. Stretch of six histidine residues interacts with nickel ion that is tightly bound to a NTA matrix   Nitrilotriacetic acid (NTA) matrix Histidine

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    GST – fusion. Principle is the same. Binds to glutation

    56

  • Слайд 57

    57 Require strong binding to glutathione GSTs function catalytically to conjugate glutathione (GSH) with a wide variety of electrophilic substrates

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    Glutathione

    58 GST from Schistosoma japonicum 1) Keeps fusion proteins soluble 2) Used for fusion purification 3) Used for protein detection with GST antibody 26 kDa tag

  • Слайд 59

    FUSION PROTEIN BOUND TO GLUTATHIONE SEPHAROSE

    59 Glutathione GST FOREIGN PEPTIDE SEPHAROSE Purification is simple : -- WASH COLUMN EXTENSIVELY -- ELUTE WITH REDUCED GLUTATHIONE -- RESULTS IN PURE GST FUSION PROTEIN

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    60

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    Some problems of production in E. coli

    61

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    Some E.coli expression host considerations

    62

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    Principal factors in bacterial expression

    63

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    Type of expression vectors

    64

  • Слайд 65

    Initiation of Transcription Promoters for Expression in Prokaryotes

    65 In Escherichia coli - Lac system - plac - Trp system - synthetic systems – ptac, ptrc In Bacillus

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    66 The Lac promoter System

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    67 The trp promoter system

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    68 E. coli Promoter Sites

  • Слайд 69

    Synthetic E. coli promoters

    69 -35 -10 ptac -> -35 box from ptrp + -10 box from plac -> pt+ac

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    70

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    71 Inverted Promoter System (from Salmonella) -> for very toxic proteins

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    Bacillus

    72 In 1872, Ferdinand Cohn, a student of Robert Koch, recognized and named the bacterium Bacillus subtilis. The organism was made to represent a large and diverse genus of Bacteria, Bacillus,  and was placed in the family Bacillaceae. The family's distinguishing feature is the production of endospores, which are highly refractile resting structures formed within the bacterial cells. Since this time, members of the genus Bacillus are characterized as Gram-positive, rod-shaped, aerobic or facultative, endospore-forming bacteria. Flagellar stains of various species of Bacillus from CDC

  • Слайд 73

    73 Antibiotic Producers: B. brevis (e.g. gramicidin, tyrothricin), B. cereus (e.g. cerexin, zwittermicin), B. circulans (e.g. circulin), B. laterosporus (e.g. laterosporin), B. licheniformis (e.g. bacitracin), B. polymyxa (e.g. polymyxin, colistin), B. pumilus (e.g. pumulin) B. subtilis (e.g. polymyxin, difficidin, subtilin, mycobacillin). Pathogens of Insects: B. larvae, B. lentimorbis, and B. popilliae are invasive pathogens. B. thuringiensis forms a parasporal crystal that is toxic to beetles. Pathogens of Animals: B. anthracis, and B. cereus.  B. alvei, B. megaterium, B. coagulans, B. laterosporus, B. subtilis, B. sphaericus, B. circulans, B. brevis, B. licheniformis, B. macerans, B. pumilus, and B. thuringiensis have been isolated from human infections. The Genus Bacillus includes two bacteria of significant medical importance, B. anthracis, the causative agent of anthrax, and B. cereus, which causes food poisoning. Nonanthrax Bacillus species can also cause a wide variety of other infections, and they are being recognized with increasing frequency as pathogens in humans.

  • Слайд 74

    74 Bacillus strains used as production organisms: - B. subtilis - B. brevis - B. licheniformis Transformation systems: - via competent cells (during transition from vegetative cells -> sporulation, cell can take up DNA (ss) when population reaches a metabolic state called competence) - protoplast - bacteriophage-mediated transduction Vectors: - replicating plasmids (pUB110, pE194, pC194, pHP13, shuttle vectors) -> replicating plasmids with temperature-sensitive origin of replication (replication stops above certain temp. -> pE194 stops above 45ºC) - integrative vectors (normally shuttle vectors) Promoters: - aprE promoter -> induction with onset of sporulation - amylase promoter -> growth-phase and nutrition regulated promoter (induction at end of exponential growth + repression by glucose) - sacB promoter (levansurase) -> not regulated - spac promoter -> hybrid promoter (subtilis phage + lac operator) -> induction with IPTG

  • Слайд 75

    Bacillus as expression host

    75

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    76

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    Products produced in Prokaryotic Systems

    77 Restriction Endonucleases -> produced in E. coli L- Ascorbic Acid (Vitamin C) -> recombinant Erwinia herbicola (gram-negative bacterium) Synthesis of Indigo (blue pigment -> dye cotton /jeans) -> produced in E. coli Amino Acids-> produced in Corynebacterium glutamicum (gram-positive bacterium) Lipases (laundry industry) -> from Pseudomonas alcaligenes produced in Pseudomonas alcaligenes Antibiotica (most of them from Streptomyces, other gram-positive bacteria, fungi) -> produced in recombinant Streptomyces and fungi (Penicillium) Biopolymers (PHB -> biodegradable plastics) -> produced in E. coli (stabilized with parB)

  • Слайд 78

    Expression in Eukaryotic Systems

    78 Yeast - Saccharomyces cerevisiae (baker’s yeast) - Pichia pastoris Insect Cells – Baculovirus Mammalian Cells

  • Слайд 79

    Expression in Yeast

    79 Autonomous replicating vectors -> shuttle vectors

  • Слайд 80

    Expression in Saccharomyces cerevisiaeAutonomous replicating systems

    80

  • Слайд 81

    Expression in Saccharomyces cerevisiaeIntegrative systems

    81 Probability for integration higher with linear fragments !

  • Слайд 82

    Expression in Saccharomyces cerevisiae

    82

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    83

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    84 Yeast are efficient secretors ! Secretory expression preferred if: -> if product toxic -> if many S-S bonds need to be closed

  • Слайд 85

    Expression in S. cerevisiae – Pichia pastoris

    85 Problems with production in S. cerevisiae: For some proteins production level low Hyperglycosylation (more than 100 mannose residues in N-glycosylation) Sometimes secretion not good -> protein stack in cells (periplasma) S. cerevisiae produces high amount of EtOH -> toxic for the cells -> effects level of production Advantages of production in Pichia pastoris: Highly efficient promoter, tightly regulated (alcohol oxidase -> AOX, induced by MeOH) Produces no EtOH -> very high cell density -> secretion very efficient Secretes very few proteins -> simplification of purification of secreted proteins

  • Слайд 86

    Expression in Pichia pastorisIntegrative systems

    86

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    Expression in Pichia pastoris

    87

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    88

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    Expression in Insect cells

    89 Baculovirus: -> infects invertebrates (insects) -> in infection cycle 2 forms of baculovirus are formed: -> single virus particle -> in protein matrix (polyhedron) trapped clusters of viruses -> during late stage of infection massive amount of polyhedron produced -> strong promoter -> polyhedron not required for virus production -> polyhedron promoter optimal for heterologous protein production in insect cells

  • Слайд 90

    90 Baculovirus: -> Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) many used as expression vector -> Production of recombinant baculovirus: 1. create a transfer vector (E. coli based plasmid with AcMNPV DNA – polyhedrin promoter/terminator + flanking sequences) -> gene of interest cloned downstream of promoter 2. Insect cells are cotransfected with virus (AcMNPV) + transfer vector -> in some double infected cells -> double crossover event (recombination) -> produce recombinant virus (bacmid -> E. coli - insect cell baculovirus shuttle vector) -> cells infected with recombinant virus -> produce plaques (lack of polyhedrin) 3. DNA hydridisation + PCR used to identify recombinant virus 4. Infection of insect cells with concentrated stock of verified recombinant virus -> 4-5 days later protein harvested

  • Слайд 91

    Baculovirus expression system

    91

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    92 Why this system? Insect cells have almost the same posttranslational modifications as mammalian cells Higher expression level than mammalian cells

  • Слайд 93

    Mammalian cell expression system

    93 1. Why do we use that system? -> to get full complement of posttranslational modifications on proteins 2. Developed cell lines: -> short term (transient) expression -> autonomous replicating systems -> viral origins (SV40) - African green monkey kidney (COS) - baby hamster kidney (BHK) - human embryonic kidney (HEK-239) -> long term (stable) expression -> integration into chromosome -> viral origins - chinese hamster ovary (CHO)

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    94

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    95  Gene expression in mammalian cell lines A convenient alternative for setting up mammalian cell facilities – get a comprehensive service from us. We will achieve stable expression of the gene of your interest in mammalian cells. Customer provides: - Mammalian vector with the gene (cDNA) to be expressed. We accept plasmid and retroviral vectors - Sequence of the gene and map of the construct for transfection Cell line or information about the cell line to be transfected. Our service includes: - Transfection of the cells. In case of a retroviral vector, virus production and cell infection - Antibiotic selection and generation of stable transfected (infected) cell clones. At least 10 independent clones will be selected and grown - Quantitative assay of the gene (cDNA) expression level in each transfected clone by RNA isolation followed by Northern hybridisation and/or RT-PCR - Selection of the best expressing clone - Cell freezing and depositing - Duration: 3-6 months (depending on the cell growth rate), allow 1month in addition if the cell line is not available in our collections Customer receives: - Detailed report on experiments and data obtained. - Two vials of transfected cells (the best expressing clone) - We will deposit the transfected cells in our collection as a precaution against accidental loss of the clone. Price guide: Price per transfection and selection of at least 10 clones: £3500.

  • Слайд 96

    Competitiveness of different expression systems

    96 http://www.proteinsciences.com/technology/pix/best_worse.gif

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