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

Сентябрь 1, 2022

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

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2. Gene Expression Systems in Prokaryotes and Eukaryotes Expression studies: 1. Analyzing Transcription - Northern blot -
3. Studying Transcription Microarray technique – DNA chips
5. Studying Transcription Primer Extension
6. Promoter Studies Used reporter genes: Lac Z GFP Luciferase Promoter
7. Promoter studies by using reporter genes
8. Luciferase (luc) systems firefly species Photinus pyralis oxidation of compounds called luciferans ( ATP-dependent process) luciferans
9. Green fluorescent protein (GFP) autofluorescent protein from Pacific Northwest jellyfish Aequorea victoria ultraviolet light causes GFP
10. GFP expression is harmless for cells and animals GFP transgenic mice from Osaka University (Masaru Okabe)
11. Engineered proteins are covering all the spectrum San Diego beach scene drawn with living bacteria expressing
12. Use of green fluorescent protein (GFP) as a reporter gene. Page 119
13. Analyzing Translation – Western Blot
14. 2 D Electrophoresis
15. Gene Expression Transcriptional start Translational start
16. Gene Expression Gene copy number: 1. Plasmid copy number: The copy-number of a plasmid in the
17. Incompatibility of plasmids: Not all plasmids are able to coexist in the same cell. Plasmids which
18. Homologous integration into chromosome Insertion on Bacillus subtilis chromosome
19. Protein expression in prokaryotic systems www.qiagen.com So, this new story would be about vectors again. Bacterial
20. General advices for one who wants to produce gene expression in prokaryotes 1. Do not forget
21. www.wzw.tum.dewww.wzw.tum.de/gene-quantification/ www.wzw.tum.de/gene-quantification/ mrna.html Introns Not an issue when you clone a cDNA
22. Orientation of insert (could go backward, if cloned with same-type sticky ends) – use incompatible sticky
23. Fusion proteins. When expressing a fusion proteins, ensure that both of them are in the same
24. PostTranslational modification Eukaryotic cells have Golgi system Prokaryotic cells do not have it nucleus Golgi
25. Efficiency of expression in E.coli Dependent of: 1. Type of transcription promoter and terminator 2. Affinity
26. Factors affecting transcription Promoters (including regulated ones) PROKARYOTIC!!!! 2. Terminators PROKARYOTIC!!!!
27. Variations between prokaryotic promoters are minimal http://www.blc.arizona.edu/marty/ 411
28. Factors affecting translation 1. Ribosome binding site (RBS) 2. Codon bias 3. Stability of the transcript
29. Ribosome binding site (RBS) = translation initiation site complimentary to 16S rRNA Avoid hairpins on 5’
30. Codon Usage in E. coli & humans
31. Codon Optimization Strategies Chemically synthesize new gene Alter sequence of the gene of interest to match
32. Commercial E. coli strains encode for a number of the rare codon genes
33. Mitochondria and chloroplast genes Alterations in the Standard Genetic Code in Mitochondria
34. Factors affecting protein stability Overall level of protease activity in bacterial cells 2. N-terminal amino acid
35. Protease-deficient host strains BL21, the work horse of E. coli expression, is deficient in two proteases
36. Inducible bacterial promoters Why not to use constitutive, always strong promoter? Induction Because recombinant (alien) protein
37. BL(DE3) inducible system and pET vectors (invented in 1984 by Bill Studier, on sale by Novagen)
38. Why repressor gene and gene of interest are expressed from different DNA molecules? Repressor gene expressed
39. Where your expressed protein will be located? Inclusion bodies (insoluble) Cytoplasm (soluble) Periplasmatic space (soluble or
40. 1. Inclusion bodies (most common case) -- Inclusion bodies are formed through the accumulation of folding
41. Electron micrograph of an inclusion body of the protein prochymosin in an E. coli cell Page
42. Good side of inclusion bodies inclusion bodies can be accumulated in the cytoplasm to much higher
43. SDS-PAGE analysis of recombinant protein produced as inclusion body hG-CSF mbel.kaist.ac.krmbel.kaist.ac.kr/research/ protein_en1.html
44. Recovery of proteins from inclusion bodies Is not a straightforward process, but road of trials and
45. Question of questions – how to purify your protein?
46. Diversity of proteins could be exploited Column chromatography Matrix particles usually packed in the column in
47. Column chromatography Different proteins are retarded to different extents by their interaction with the matrix, they
48. (A) ION-EXCHANGE CHROMATOGRAPHY Ion-exchange columns are packed with small beads that carry positive or negative charges
49. (B) GEL-FILTRATION CHROMATOGRAPHY Gel-filtration columns separate proteins according to their size on tiny porous beads. Protein
50. (C) AFFINITY CHROMATOGRAPHY Affinity columns contain a matrix covalently coupled to a molecule that interacts specifically
51. Protein electrophoresis Essential Cell Biology: An Introduction to the Molecular Biology of the Cell
52. www.unizh.chwww.unizh.ch/.../www.unizh.ch/.../Teaching_slide_showswww.unizh.ch/.../Teaching_slide_shows/ Lambda/sld015.htm www.unizh.chwww.unizh.ch/.../www.unizh.ch/.../Teaching_slide_showswww.unizh.ch/.../Teaching_slide_shows/ Lambda/sld015.htm
53. Fusion proteins increase production level facilitate purification (taq) detection of expression (GFP fusion) Redirection of proteins
54. Most widely used purification strategy – to produce your protein as a fusion with something easily
55. Histidine: a charged aminoacid The affinity of this interaction is very high which allows protein purification
56. GST – fusion. Principle is the same. Binds to glutation
57. Require strong binding to glutathione Require strong binding to glutathione GSTs function catalytically to conjugate glutathione
58. Glutathione GST from Schistosoma japonicum 1) Keeps fusion proteins soluble 2) Used for fusion purification 3)
59. FUSION PROTEIN BOUND TO GLUTATHIONE SEPHAROSE Glutathione GST FOREIGN PEPTIDE SEPHAROSE Purification is simple : --
61. Some problems of production in E. coli
62. Some E.coli expression host considerations
63. Principal factors in bacterial expression
64. Type of expression vectors
65. Initiation of Transcription Promoters for Expression in Prokaryotes In Escherichia coli - Lac system - plac
66. The Lac promoter System
67. The trp promoter system
68. E. coli Promoter Sites
69. Synthetic E. coli promoters -35 -10 ptac -> -35 box from ptrp + -10 box from
71. Inverted Promoter System (from Salmonella) -> for very toxic proteins
72. Bacillus In 1872, Ferdinand Cohn, a student of Robert Koch, recognized and named the bacterium Bacillus
73. Bacillus Antibiotic Producers: B. brevis (e.g. gramicidin, tyrothricin), B. cereus (e.g. cerexin, zwittermicin), B. circulans (e.g.
74. Bacillus Bacillus strains used as production organisms: - B. subtilis - B. brevis - B. licheniformis
75. Bacillus as expression host
76. Bacillus as expression host
77. Products produced in Prokaryotic Systems Restriction Endonucleases -> produced in E. coli L- Ascorbic Acid (Vitamin
78. Expression in Eukaryotic Systems Yeast - Saccharomyces cerevisiae (baker’s yeast) - Pichia pastoris Insect Cells –
79. Expression in Yeast Autonomous replicating vectors -> shuttle vectors
80. Expression in Saccharomyces cerevisiae Autonomous replicating systems
81. Expression in Saccharomyces cerevisiae Integrative systems Probability for integration higher with linear fragments !
82. Expression in Saccharomyces cerevisiae
83. Expression in Saccharomyces cerevisiae
84. Yeast are efficient secretors ! Secretory expression preferred if: -> if product toxic -> if many
85. Expression in S. cerevisiae – Pichia pastoris Problems with production in S. cerevisiae: For some proteins
86. Expression in Pichia pastoris Integrative systems
87. Expression in Pichia pastoris
88. Expression in Pichia pastoris
89. Expression in Insect cells Baculovirus: -> infects invertebrates (insects) -> in infection cycle 2 forms of
90. Expression in Insect cells Baculovirus: -> Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) many used as
91. Baculovirus expression system
92. Why this system? Insect cells have almost the same posttranslational modifications as mammalian cells Higher expression
93. Mammalian cell expression system 1. Why do we use that system? -> to get full complement
94. Mammalian cell expression system
95. Gene expression in mammalian cell lines A convenient alternative for setting up mammalian cell facilities –
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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

Слайд 3

Studying Transcription
Microarray technique – DNA chips

Слайд 4

Слайд 5

Studying Transcription
Primer Extension

Слайд 6

Promoter Studies
Used reporter genes:
Lac Z
GFP
Luciferase
Promoter

Слайд 7

Promoter studies by using reporter genes

Слайд 8

Luciferase (luc) systems
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)
autofluorescent protein from Pacific Northwest jellyfish
Aequorea victoria
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 harmless for cells and animals
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

Engineered proteins
are covering
all the spectrum
San Diego beach scene
drawn

with living bacteria
expressing 8 different colors
of fluorescent proteins.

Many more fluorescent proteins are engineered

Слайд 12

Use of green fluorescent protein (GFP) as a reporter gene.
Page 119

Слайд 13

Analyzing Translation – Western Blot

Слайд 14

2 D Electrophoresis

Слайд 15

Gene Expression
Transcriptional start
Translational start

Слайд 16

Gene Expression
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

Слайд 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

Слайд 18

Homologous integration into chromosome
Insertion on Bacillus subtilis chromosome

Слайд 19

Protein expression in prokaryotic systems
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

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

www.wzw.tum.dewww.wzw.tum.de/gene-quantification/ www.wzw.tum.de/gene-quantification/ mrna.html
Introns
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

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

Слайд 23

Fusion proteins.
When expressing
a fusion proteins,
ensure that
both of them

are
in the same reading frame

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

Слайд 24

PostTranslational modification
Eukaryotic cells have Golgi system
Prokaryotic cells do not

have it

nucleus

Golgi

Слайд 25

Efficiency of expression in E.coli
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
Promoters (including regulated ones)
PROKARYOTIC!!!!
2. Terminators
PROKARYOTIC!!!!

Слайд 27

Variations between prokaryotic promoters are minimal
http://www.blc.arizona.edu/marty/
411

Слайд 28

Factors affecting translation
1. Ribosome binding site (RBS)
2. Codon bias
3. Stability of

the transcript

Слайд 29

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

rRNA

<10 nt

Avoid hairpins
on 5’ end of gene
(minimize GC content)

Examining the second codon;
better AAA – lysin (13.9% of all E.coli genes).
Expression can vary 15 times.

Слайд 30

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

Слайд 32

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

codon genes

Слайд 33

Mitochondria and chloroplast genes
Alterations in the Standard Genetic Code in

Mitochondria

Слайд 34

Factors affecting protein stability
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 proline E glutamic acid S serine T threonine

…. Mutate PEST aminoacids….

Слайд 35

Protease-deficient host strains
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
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)

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?

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?
Inclusion bodies
(insoluble)
Cytoplasm
(soluble)
Periplasmatic

space
(soluble or insoluble)

Secreted (!!)

E.Coli
can not do that

Слайд 40

1. Inclusion bodies (most common case)
-- 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

Page 116

Protein Folding

Слайд 42

Good side of inclusion bodies
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 are resistant to proteolysis
That results in the high yield of protein production.

Слайд 43

SDS-PAGE analysis of recombinant protein produced as inclusion body
hG-CSF
mbel.kaist.ac.krmbel.kaist.ac.kr/research/

protein_en1.html

Слайд 44

Recovery of proteins from inclusion bodies
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?

Слайд 46

Diversity of proteins could be exploited
Column chromatography
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.