Cell-free Bacterial Yeast Insect Mammalian Protein Expression Systems.
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Transcript of Cell-free Bacterial Yeast Insect Mammalian Protein Expression Systems.
Cell-free
Bacterial
Yeast
Insect
Mammalian
Protein Expression Systems
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
RBS RBS 5-9 n STARTGAAGGAATTCAGGAGCCCTTCACCATG ... ...
Ribosome Bindind Site (RBS):
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 promoters2500 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)
Key features
Plac Low level up to middle (IPTG)
Weak, regulated. Suitable for expression of gene products at very low intracellular level. Comparatively expensive induction.
Ptac
Ptrc
(trp-lac)
Moderately high (IPTG)
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
T5
Very high
High
Utilizes T7 RNA polymerase.
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 BacteriaPart2
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 EnzymesNcoI
HindIII
Cloning Using A-overhangs
TA-Cloning with Topoisomerase
Directional Cloning
CACC
Gateway Technology
Expression of Fusion ProteinsWe 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 ConstructionNcoI HindIII
HindIII
PstI
pUC18/19
pUC182686 bp
APr
ALPHA
P(BLA)
P(LAC)
ORI
AvaI (435)
BamHI (430)
EcoRI (451)
HindIII (400)
PstI (416)
SmaI (437)
XmaI (435)
XbaI (424)
ApaLI (178)
ApaLI (1121)
ApaLI (2367)
Transcriptional vector
pTrc99
Translational vector
pQE
Translational vector + CDR
pET
pCR&pEXP
pBAD
Expression strains
# Strain Key features 1 BL21 Deficient in lon and ompT proteases 2 BL21* /STAR #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 #1 + carry T7 polymerase under PPBAD (araBAD) Enables T7 expression with tight regulation
6 BL21 CodonPlus-RIL
#1 + Enhances the expression of eukaryotic proteins that contain codons rarely used in E. coli: AGG, AGA, AUA, CUA
7 BL21 trxB #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)