Презентация на тему Gene Expression Systems in Prokaryotes and Eukaryotes

1. Analyzing Transcription
- Northern

Luciferase
Promoter

compounds called luciferans
( ATP-dependent process)
luciferans emit fluorescense
Expressed luciferase catalyses


mouse

Mice are injected
with LUC+ salmonellas.
Sensitive digital cameras
allow non-invasive detection.
For GT vectors
pics look the same

luminometer measurement

jellyfish
Aequorea victoria



ultraviolet light causes GFP
to autofluoresce
In

Jellyfish do nothing with UV,
The activate GFP by aequorin
(Ca++ activated,
biolumuniscent helper)

transgenic mice from
Osaka University
(Masaru Okabe)
GFP construct could

GFP labelled image of a human tumor.
Vessel on the tumor surface
are visible in black

beach scene
drawn with living bacteria
expressing 8 different

Many more fluorescent proteins are engineered

reporter gene.
Page 119

number:
The copy-number of a plasmid in

coexist in the same cell.
Plasmids which have the

would be about vectors again.
Bacterial expression vectors
have

Inducible promoter systems;

Protein fusions including fused tags;

gene expression in prokaryotes
1. Do not forget to cut

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

a cDNA

with same-type sticky ends) – use incompatible sticky ends
www.bch.bris.ac.ukwww.bch.bris.ac.uk/staff/

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/

cells do not have it
nucleus
Golgi

of transcription promoter and terminator
2. Affinity of mRNA and

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

Terminators
PROKARYOTIC!!!!

bias
3. Stability of the transcript

complimentary to 16S rRNA

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

the gene of interest
to match donor codons to

of the rare codon genes

Genetic Code in Mitochondria

activity
in bacterial cells
2. N-terminal amino acid affects

3. Internal regions containing clusters of certain amino acids
can increase proteolysis

P proline E glutamic acid S serine T threonine

…. Mutate PEST aminoacids….

E. coli expression,
is deficient in two proteases
encoded

It is dangerous
to kill proteases,
it makes E.coli
grow much slowly
as proteases needed
for proper metabolism

strong promoter?






















Induction
Because recombinant (alien) protein
is often

Bacterial grow takes time….

1984 by Bill Studier, on sale by Novagen)
1) T7

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

expressed from different DNA molecules?
Repressor gene expressed from

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)

bodies
(insoluble)
Cytoplasm
(soluble)
Periplasmatic space
(soluble or insoluble)
Secreted (!!)
E.Coli
can

bodies are formed through the accumulation
of folding intermediates

-- 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...

protein prochymosin in an E. coli cell
Page 116
Protein Folding

accumulated in the cytoplasm
to much higher level (greater

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.

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

straightforward process, but road of trials and errors
Solubilization
Refolding
Choice of

-- 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)

protein?

particles
usually packed in the column
in the form

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

by their interaction with the matrix,
they can be

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

beads
that carry
positive or negative charges
retarding proteins
of

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

their size
on tiny porous beads.

Protein molecules
that are

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

to a molecule that interacts specifically
with the protein

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

the Molecular Biology of the Cell

(GFP fusion)
Redirection of proteins (secretion -> signal peptidases)
Surface display

your protein as a fusion with something easily purifyable

(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)

interaction is very high
which allows protein purification to

Stretch of six histidine residues
interacts with nickel ion
that is tightly bound to a NTA matrix  

Nitrilotriacetic acid (NTA) matrix

Histidine

to glutation

to glutathione
GSTs function catalytically to conjugate glutathione (GSH)

soluble
2) Used for fusion purification
3) Used for protein

26 kDa tag

simple :

-- WASH COLUMN EXTENSIVELY

-- ELUTE WITH REDUCED

Prokaryotes
In Escherichia coli

- Lac system - plac

ptrp + -10 box from plac -> pt+ac

toxic proteins

Koch, recognized and named the bacterium Bacillus subtilis.
The

Flagellar stains of various species of Bacillus from CDC

cereus (e.g. cerexin, zwittermicin), B. circulans (e.g. circulin), B.

- B. subtilis

produced in E. coli
L- Ascorbic Acid (Vitamin C) ->

(baker’s yeast)
- Pichia pastoris

Insect Cells – Baculovirus

with linear fragments !

if product toxic
-> if many S-S bonds need to

production in S. cerevisiae:

For some proteins production level low
Hyperglycosylation

infection cycle 2 forms of baculovirus are formed:

polyhedrosis virus (AcMNPV) many used as expression vector

-> Production

posttranslational modifications as mammalian cells
Higher expression level than mammalian

Baculovirus expression system

that system?
-> to get full complement of

for setting up mammalian cell facilities – get a