Transcript Protein expression in eukaryotic hosts

Protein Expression Systems
Bacterial
Cell-free
Yeast
Mammalian
Insect
Protein Expression in Bacteria
1. Advantages/disadvantages
2. Genetic elements essential for the expression
3. Cloning strategies
4. Overview of the available expression systems
and expression strains
5. Design of cloning procedures using the VNTI
program
Advantages
• Fast growth
• Cheap medium and equipment for growing
• Good knowledge of the host
Disadvantages
Limitation for expression of eukaryotic proteins due to:
•
different frequencies with which the different
codons appear in genes of these organisms
E.g. CGT, CGC, CGG, AGG, AGA, CGA code for arginine,
but the last 3 (AGG, AGA, CGA) are rarely used in E. coli
and it has low amounts of respective tRNAs.
•
differences in post-translational modifications
(SS bonds, glycosylation etc)
Disadvantages
Accumulation of lipopolysaccharides
(generally referred to as endotoxins) …
Goals
To obtain
as much as possible
/good expression+good cell growth
soluble folded protein
/reduced aggregation
in a form that is easy to purify
/use of secretion and tags
Common problem:
High expression=danger of aggregation, decreased cell growth
Genetic Elements Essential for Expression
RBS, START, and STOP
Ribosome Bindind Site (RBS):
RBS
RBS
5-9 n START
GAAGGAATTCAGGAGCCCTTCACCATG ... ...
START codons:
E. coli uses 77% ATG (AUG), 14% GTG (GUG), 8%
TTG (UUG) and a few others
STOP codons:
TAG (UAG), TGA (UGA), TAA (UAA)
Genetic Elements Essential for Expression
Promoters
Host’s promoters
2500 in the entire genome of E. coli K12 strain
Most frequently used: Plac / Ptac / Ptrc, PPBAD, rhaPBAD
- Regulation of expression
Promoters from phages
T7, T3, SP6, T5, PL
- Highly efficient and specific expression
Plac: Regulation
Plac, Ptac, Ptrc: Characteristics
Level of expression
(inductor)
Plac
Low level up to
middle (IPTG)
Ptac
Moderately high
(IPTG)
Ptrc
(trp-lac)
Key features
Weak, regulated. Suitable for
expression of gene products at
very low intracellular level.
Comparatively expensive
induction.
High-level, but lower than T7
system. Regulated expression still
possible.Comparatively expensive
induction. High basal level.
PPBAD: Regulation
PPBAD and RhaPBAD
Level of expression
(inductor)
Key features
PPBAD
Variable from low to
high level
(L-arabinose)
Can fine-tune expression levels in
a dose-dependent manner. Tight
regulation possible. Low basal
level. Inexpensive inducer.
rhaPBAD
Variable from low to
high level
(L-rhamnose)
Tight regulation. Low basal
activity. Relatively expensive
inducer.
Phage Promoters
Level of expression
(inductor)
Key features
T7
Very high
Utilizes T7 RNA polymerase.
T5
High
Utilizes E. coli RNA polymerase.
PL
Moderately high
(temperature shift)
Temperature-sensitive host required.
Less likelihood of "leaky" un-induced
expression. Basal level; high basal
level by temperatures below 30°C.
No inducer.
Combinations
Genetic Elements Essential for Expression
Replication Origin
Plasmid
Replicon
Copy Number
pBR322
pMB1
15-20
pUC
pUC
500-700
pACYC
p15A
18-22
pSC101
pSC101
5
colE1
colE1
15-20
Co-expression from two plasmids
Protein Expression in Bacteria
Part2
1. Cloning strategies
2. Overview of the available expression
systems and expression strains
3. Design of cloning procedures using the
VNTI program
Types of Expression Vectors
1
2
3
Insertion into Transcriptional Vectors
Insertion into Translational Vectors
Cloning Using Restriction Enzymes
NcoI
HindIII
Cloning Using A-overhangs
TA-Cloning with Topoisomerase
Directional Cloning
CACC
Gateway Technology
Expression of Fusion Proteins
We may fuse the target protein with
•
various tags to facilitate its purification or detection
HHHHHH-target, epitope-target
•
highly soluble proteins to improve solubility and to
facilitate purification
Thioredoxin-target, GST-target
•
signal peptides or other proteins or domains to
promote secretion
SP-target
‘Short’ Fusion Protein Construction
ATG CAT CAC CAT CAC CAT CAC
‘Long’ Fusion Protein Construction
NcoI
HindIII
HindIII
PstI
pUC18/19
ApaLI (178)
ALPHA
P(BLA)
HindIII (400)
ApaLI (2367)
PstI (416)
XbaI (424)
BamHI (430)
AvaI (435)
APr
pUC18
2686 bp
XmaI (435)
SmaI (437)
EcoRI (451)
P(LAC)
ORI
ApaLI (1121)
Transcriptional vector
pTrc99
Translational vector
pQE
Translational vector + CDR
pET
pCR&pEXP
pBAD
Expression strains
# Strain
1 BL21
2 BL21* /STAR
Key features
Deficient in lon and ompT proteases
#1 + deficient in RNaseE
Improves the stability of mRNA transcripts and increases
protein expression yield
3 BL21*(DE3)
#1 or 2 + carry T7 polymerase under Plac
Enables T7 expression
4 BL21*(DE3)pLysS/E #3 + plasmid pLysS or pLysE expressing T7 lysozyme
Reduces basal expression of recombinant genes
5 BL21-AI
6 BL21 CodonPlusRIL
7 BL21 trxB
#1 + carry T7 polymerase under PPBAD (araBAD)
Enables T7 expression with tight regulation
#1 + Enhances the expression of eukaryotic proteins that
contain codons rarely used in E. coli: AGG, AGA, AUA, CUA
#1 + deficient of trxB
Facilitates cytoplasmic disulfide bond formation
Expression optimization
To optimase:
Level of inducer (e.g. arabinose)
Time of induction
Temperature of the induction step (popular - 18oC
overnight)