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Презентация на тему: Gene Expression Systems in Prokaryotes and Eukaryotes


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Презентация на тему: Gene Expression Systems in Prokaryotes and Eukaryotes


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№ слайда 1 Gene Expression Systems in Prokaryotes and Eukaryotes Expression studies Express
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Gene Expression Systems in Prokaryotes and Eukaryotes Expression studies Expression in Prokaryotes (Bacteria) Expression in Eukaryotes

№ слайда 2 Gene Expression Systems in Prokaryotes and Eukaryotes Expression studies: 1. Ana
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Gene Expression Systems in Prokaryotes and Eukaryotes 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

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№ слайда 6 Promoter Studies
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Promoter Studies

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№ слайда 8 Luciferase (luc) systems
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Luciferase (luc) systems

№ слайда 9 Green fluorescent protein (GFP)
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Green fluorescent protein (GFP)

№ слайда 10 GFP expression is harmless for cells and animals
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GFP expression is harmless for cells and animals

№ слайда 11 Many more fluorescent proteins are engineered
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Many more fluorescent proteins are engineered

№ слайда 12 Use of green fluorescent protein (GFP) as a reporter gene.
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Use of green fluorescent protein (GFP) as a reporter gene.

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№ слайда 14 2 D Electrophoresis
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2 D Electrophoresis

№ слайда 15 Gene Expression
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Gene Expression

№ слайда 16 Gene Expression
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Gene Expression

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№ слайда 18 Homologous integration into chromosome
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Homologous integration into chromosome

№ слайда 19 Protein expression in prokaryotic systems
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Protein expression in prokaryotic systems

№ слайда 20 General advices for one who wants to produce gene expression in prokaryotes
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General advices for one who wants to produce gene expression in prokaryotes

№ слайда 21 Introns
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Introns

№ слайда 22 Orientation of insert (could go backward, if cloned with same-type sticky ends)
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Orientation of insert (could go backward, if cloned with same-type sticky ends) – use incompatible sticky ends

№ слайда 23 Fusion proteins.
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Fusion proteins.

№ слайда 24 PostTranslational modification
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PostTranslational modification

№ слайда 25 Efficiency of expression in E.coli
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Efficiency of expression in E.coli

№ слайда 26 Factors affecting transcription
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Factors affecting transcription

№ слайда 27 Variations between prokaryotic promoters are minimal
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Variations between prokaryotic promoters are minimal

№ слайда 28 Factors affecting translation
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Factors affecting translation

№ слайда 29 Ribosome binding site (RBS) = translation initiation site complimentary to 16S r
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Ribosome binding site (RBS) = translation initiation site complimentary to 16S rRNA

№ слайда 30 Codon Usage in E. coli & humans
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Codon Usage in E. coli & humans

№ слайда 31 Codon Optimization Strategies Chemically synthesize new gene Alter sequence of t
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Codon Optimization Strategies 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

№ слайда 32 Commercial E. coli strains encode for a number of the rare codon genes
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Commercial E. coli strains encode for a number of the rare codon genes

№ слайда 33 Mitochondria and chloroplast genes
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Mitochondria and chloroplast genes

№ слайда 34 Factors affecting protein stability
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Factors affecting protein stability

№ слайда 35 Protease-deficient host strains
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Protease-deficient host strains

№ слайда 36 Inducible bacterial promoters
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Inducible bacterial promoters

№ слайда 37 BL(DE3) inducible system and pET vectors (invented in 1984 by Bill Studier, on s
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BL(DE3) inducible system and pET vectors (invented in 1984 by Bill Studier, on sale by Novagen)

№ слайда 38 Why repressor gene and gene of interest are expressed from different DNA molecul
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Why repressor gene and gene of interest are expressed from different DNA molecules?

№ слайда 39 Where your expressed protein will be located?
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Where your expressed protein will be located?

№ слайда 40 1. Inclusion bodies (most common case)
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1. Inclusion bodies (most common case)

№ слайда 41 Electron micrograph of an inclusion body of the protein prochymosin in an E. col
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Electron micrograph of an inclusion body of the protein prochymosin in an E. coli cell

№ слайда 42 Good side of inclusion bodies
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Good side of inclusion bodies

№ слайда 43 SDS-PAGE analysis of recombinant protein produced as inclusion body
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SDS-PAGE analysis of recombinant protein produced as inclusion body

№ слайда 44 Recovery of proteins from inclusion bodies
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Recovery of proteins from inclusion bodies

№ слайда 45 Question of questions – how to purify your protein?
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Question of questions – how to purify your protein?

№ слайда 46 Diversity of proteins could be exploited
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Diversity of proteins could be exploited

№ слайда 47 Column chromatography
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Column chromatography

№ слайда 48 (A) ION-EXCHANGE CHROMATOGRAPHY
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(A) ION-EXCHANGE CHROMATOGRAPHY

№ слайда 49 (B) GEL-FILTRATION CHROMATOGRAPHY
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(B) GEL-FILTRATION CHROMATOGRAPHY

№ слайда 50 (C) AFFINITY CHROMATOGRAPHY
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(C) AFFINITY CHROMATOGRAPHY

№ слайда 51 Protein electrophoresis
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Protein electrophoresis

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№ слайда 53 Fusion proteins increase production level facilitate purification (taq) detectio
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Fusion proteins 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,..)

№ слайда 54 Most widely used purification strategy – to produce your protein as a fusion wit
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Most widely used purification strategy – to produce your protein as a fusion with something easily purifyable

№ слайда 55 Histidine: a charged aminoacid
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Histidine: a charged aminoacid

№ слайда 56 GST – fusion. Principle is the same. Binds to glutation
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GST – fusion. Principle is the same. Binds to glutation

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№ слайда 58 Glutathione
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Glutathione

№ слайда 59 FUSION PROTEIN BOUND TO GLUTATHIONE SEPHAROSE
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FUSION PROTEIN BOUND TO GLUTATHIONE SEPHAROSE

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№ слайда 61 Some problems of production in E. coli
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Some problems of production in E. coli

№ слайда 62 Some E.coli expression host considerations
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Some E.coli expression host considerations

№ слайда 63 Principal factors in bacterial expression
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Principal factors in bacterial expression

№ слайда 64 Type of expression vectors
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Type of expression vectors

№ слайда 65 Initiation of Transcription Promoters for Expression in Prokaryotes In Escherich
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Initiation of Transcription Promoters for Expression in Prokaryotes In Escherichia coli - Lac system - plac - Trp system - synthetic systems – ptac, ptrc In Bacillus

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№ слайда 69 Synthetic E. coli promoters
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Synthetic E. coli promoters

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№ слайда 72 Bacillus
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Bacillus

№ слайда 73 Bacillus Antibiotic Producers: B. brevis (e.g. gramicidin, tyrothricin), B. cere
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Bacillus 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.

№ слайда 74 Bacillus Bacillus strains used as production organisms: - B. subtilis - B. brevi
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Bacillus 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

№ слайда 75 Bacillus as expression host
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Bacillus as expression host

№ слайда 76 Bacillus as expression host
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Bacillus as expression host

№ слайда 77 Products produced in Prokaryotic Systems Restriction Endonucleases -> produce
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Products produced in Prokaryotic Systems 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)

№ слайда 78 Expression in Eukaryotic Systems Yeast - Saccharomyces cerevisiae (baker’s yeast
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Expression in Eukaryotic Systems Yeast - Saccharomyces cerevisiae (baker’s yeast) - Pichia pastoris Insect Cells – Baculovirus Mammalian Cells

№ слайда 79 Expression in Yeast
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Expression in Yeast

№ слайда 80 Expression in Saccharomyces cerevisiae Autonomous replicating systems
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Expression in Saccharomyces cerevisiae Autonomous replicating systems

№ слайда 81 Expression in Saccharomyces cerevisiae Integrative systems
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Expression in Saccharomyces cerevisiae Integrative systems

№ слайда 82 Expression in Saccharomyces cerevisiae
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Expression in Saccharomyces cerevisiae

№ слайда 83 Expression in Saccharomyces cerevisiae
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Expression in Saccharomyces cerevisiae

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№ слайда 85 Expression in S. cerevisiae – Pichia pastoris Problems with production in S. cer
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Expression in S. cerevisiae – Pichia pastoris 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

№ слайда 86 Expression in Pichia pastoris Integrative systems
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Expression in Pichia pastoris Integrative systems

№ слайда 87 Expression in Pichia pastoris
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Expression in Pichia pastoris

№ слайда 88 Expression in Pichia pastoris
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Expression in Pichia pastoris

№ слайда 89 Expression in Insect cells Baculovirus: -> infects invertebrates (insects) -&
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Expression in Insect cells 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

№ слайда 90 Expression in Insect cells Baculovirus: -> Autographa californica multiple nu
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Expression in Insect cells 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

№ слайда 91 Baculovirus expression system
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Baculovirus expression system

№ слайда 92 Baculovirus expression system Why this system? Insect cells have almost the same
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Baculovirus expression system Why this system? Insect cells have almost the same posttranslational modifications as mammalian cells Higher expression level than mammalian cells

№ слайда 93 Mammalian cell expression system 1. Why do we use that system? -> to get full
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Mammalian cell expression system 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)

№ слайда 94 Mammalian cell expression system
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Mammalian cell expression system

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№ слайда 96 Competitiveness of different expression systems
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Competitiveness of different expression systems

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