🗊 Gene Expression Systems in Prokaryotes and Eukaryotes Expression studies Expression in Prokaryotes (Bacteria) Expression in Eukaryotes

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  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №1  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №2  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №3  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №4  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №5  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №6  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №7  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №8  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №9  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №10  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №11  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №12  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №13  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №14  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №15  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №16  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №17  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №18  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №19  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №20  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №21  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №22  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №23  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №24  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №25  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №26  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №27  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №28  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №29  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №30  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №31  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №32  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №33  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №34  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №35  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №36  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №37  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №38  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №39  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №40  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №41  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №42  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №43  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №44  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №45  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №46  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №47  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №48  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №49  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №50  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №51  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №52  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №53  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №54  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №55  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №56  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №57  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №58  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №59  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №60  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №61  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №62  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №63  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №64  
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  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №71  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №72  
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  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №74  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №75  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №76  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №77  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №78  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №79  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №80  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №81  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №82  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №83  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №84  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №85  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №86  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №87  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №88  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №89  
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  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №92  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №93  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №94  
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Слайды и текст этой презентации

Слайд 1



Gene Expression Systems in Prokaryotes and Eukaryotes
Expression studies
Expression in Prokaryotes (Bacteria)
Expression in Eukaryotes
Описание слайда:
Gene Expression Systems in Prokaryotes and Eukaryotes Expression studies Expression in Prokaryotes (Bacteria) Expression in Eukaryotes

Слайд 2



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
Описание слайда:
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

Слайд 3
  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №3
Описание слайда:

Слайд 4
  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №4
Описание слайда:

Слайд 5
  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №5
Описание слайда:

Слайд 6



Promoter Studies
Описание слайда:
Promoter Studies

Слайд 7
  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №7
Описание слайда:

Слайд 8



Luciferase (luc) systems
Описание слайда:
Luciferase (luc) systems

Слайд 9



Green fluorescent protein (GFP)
Описание слайда:
Green fluorescent protein (GFP)

Слайд 10



GFP expression is harmless
for cells and animals
Описание слайда:
GFP expression is harmless for cells and animals

Слайд 11



Many more fluorescent proteins are engineered
Описание слайда:
Many more fluorescent proteins are engineered

Слайд 12



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

Слайд 13
  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №13
Описание слайда:

Слайд 14



2 D Electrophoresis
Описание слайда:
2 D Electrophoresis

Слайд 15



Gene Expression
Описание слайда:
Gene Expression

Слайд 16



Gene Expression
Описание слайда:
Gene Expression

Слайд 17
  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №17
Описание слайда:

Слайд 18



Homologous integration into chromosome
Описание слайда:
Homologous integration into chromosome

Слайд 19



Protein expression in prokaryotic systems
Описание слайда:
Protein expression in prokaryotic systems

Слайд 20



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

Слайд 21



Introns
Описание слайда:
Introns

Слайд 22



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

Слайд 23



Fusion proteins.
Описание слайда:
Fusion proteins.

Слайд 24



PostTranslational modification
Описание слайда:
PostTranslational modification

Слайд 25



Efficiency of expression in E.coli
Описание слайда:
Efficiency of expression in E.coli

Слайд 26



Factors affecting transcription
Описание слайда:
Factors affecting transcription

Слайд 27



Variations between prokaryotic promoters are minimal
Описание слайда:
Variations between prokaryotic promoters are minimal

Слайд 28



Factors affecting translation
Описание слайда:
Factors affecting translation

Слайд 29



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

Слайд 30



Codon Usage in E. coli & humans
Описание слайда:
Codon Usage in E. coli & humans

Слайд 31



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
Описание слайда:
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

Слайд 32



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

Слайд 33



Mitochondria and chloroplast genes
Описание слайда:
Mitochondria and chloroplast genes

Слайд 34



Factors affecting protein stability
Описание слайда:
Factors affecting protein stability

Слайд 35



Protease-deficient host strains
Описание слайда:
Protease-deficient host strains

Слайд 36



Inducible bacterial promoters
Описание слайда:
Inducible bacterial promoters

Слайд 37



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

Слайд 38



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

Слайд 39



Where your expressed protein will be located?
Описание слайда:
Where your expressed protein will be located?

Слайд 40



1. Inclusion bodies 
(most common case)
Описание слайда:
1. Inclusion bodies (most common case)

Слайд 41



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

Слайд 42



Good side of inclusion bodies
Описание слайда:
Good side of inclusion bodies

Слайд 43



SDS-PAGE analysis of recombinant protein produced as inclusion body
Описание слайда:
SDS-PAGE analysis of recombinant protein produced as inclusion body

Слайд 44



Recovery of proteins from inclusion bodies
Описание слайда:
Recovery of proteins from inclusion bodies

Слайд 45



Question of questions – 
how to purify your protein?
Описание слайда:
Question of questions – how to purify your protein?

Слайд 46



Diversity of proteins could be exploited
Описание слайда:
Diversity of proteins could be exploited

Слайд 47



Column chromatography
Описание слайда:
Column chromatography

Слайд 48



(A) ION-EXCHANGE CHROMATOGRAPHY
Описание слайда:
(A) ION-EXCHANGE CHROMATOGRAPHY

Слайд 49



(B) GEL-FILTRATION CHROMATOGRAPHY
Описание слайда:
(B) GEL-FILTRATION CHROMATOGRAPHY

Слайд 50



(C) AFFINITY CHROMATOGRAPHY
Описание слайда:
(C) AFFINITY CHROMATOGRAPHY

Слайд 51



Protein 
electrophoresis
Описание слайда:
Protein electrophoresis

Слайд 52
  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №52
Описание слайда:

Слайд 53



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,..)
Описание слайда:
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,..)

Слайд 54



Most widely used purification strategy – to produce your protein as a fusion 
with something easily purifyable
Описание слайда:
Most widely used purification strategy – to produce your protein as a fusion with something easily purifyable

Слайд 55



Histidine: a charged aminoacid
Описание слайда:
Histidine: a charged aminoacid

Слайд 56



GST – fusion. Principle is the same. Binds to glutation
Описание слайда:
GST – fusion. Principle is the same. Binds to glutation

Слайд 57
  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №57
Описание слайда:

Слайд 58



Glutathione
Описание слайда:
Glutathione

Слайд 59



FUSION PROTEIN BOUND TO GLUTATHIONE SEPHAROSE
Описание слайда:
FUSION PROTEIN BOUND TO GLUTATHIONE SEPHAROSE

Слайд 60
  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №60
Описание слайда:

Слайд 61



Some problems of production in E. coli
Описание слайда:
Some problems of production in E. coli

Слайд 62



Some E.coli expression host considerations
Описание слайда:
Some E.coli expression host considerations

Слайд 63



Principal factors in bacterial expression
Описание слайда:
Principal factors in bacterial expression

Слайд 64



Type of expression vectors
Описание слайда:
Type of expression vectors

Слайд 65



Initiation of Transcription  
 Promoters for Expression in Prokaryotes
In Escherichia coli
    - Lac system - plac
    - Trp system
    - synthetic systems – ptac, ptrc
In Bacillus
Описание слайда:
Initiation of Transcription Promoters for Expression in Prokaryotes In Escherichia coli - Lac system - plac - Trp system - synthetic systems – ptac, ptrc In Bacillus

Слайд 66
  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №66
Описание слайда:

Слайд 67
  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №67
Описание слайда:

Слайд 68
  
  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №68
Описание слайда:

Слайд 69



Synthetic E. coli promoters
Описание слайда:
Synthetic E. coli promoters

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  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №70
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  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №71
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Bacillus
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Bacillus

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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.
Описание слайда:
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.

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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
Описание слайда:
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

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Bacillus as expression host
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Bacillus as expression host

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Bacillus as expression host
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Bacillus as expression host

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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)
Описание слайда:
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)

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Expression in Eukaryotic Systems
Yeast
    - Saccharomyces cerevisiae (baker’s yeast)
    - Pichia pastoris
Insect Cells – Baculovirus 
Mammalian Cells
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Expression in Eukaryotic Systems Yeast - Saccharomyces cerevisiae (baker’s yeast) - Pichia pastoris Insect Cells – Baculovirus Mammalian Cells

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Expression in Yeast
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Expression in Yeast

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Expression in Saccharomyces cerevisiae
Autonomous replicating systems
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Expression in Saccharomyces cerevisiae Autonomous replicating systems

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Expression in Saccharomyces cerevisiae
Integrative systems
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Expression in Saccharomyces cerevisiae Integrative systems

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Expression in Saccharomyces cerevisiae
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Expression in Saccharomyces cerevisiae

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Expression in Saccharomyces cerevisiae
Описание слайда:
Expression in Saccharomyces cerevisiae

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  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №84
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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
Описание слайда:
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

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Expression in Pichia pastoris
Integrative systems
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Expression in Pichia pastoris Integrative systems

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Expression in Pichia pastoris
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Expression in Pichia pastoris

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Expression in Pichia pastoris
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Expression in Pichia pastoris

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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
Описание слайда:
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

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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
Описание слайда:
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

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Baculovirus expression system
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Baculovirus expression system

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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
Описание слайда:
Baculovirus expression system Why this system? Insect cells have almost the same posttranslational modifications as mammalian cells Higher expression level than mammalian cells

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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)
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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)

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Mammalian cell expression system
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Mammalian cell expression system

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  Gene Expression Systems in Prokaryotes and Eukaryotes  Expression studies  Expression in Prokaryotes (Bacteria)  Expression in Eukaryotes  , слайд №95
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Competitiveness 
of different expression systems
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Competitiveness of different expression systems



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