Lecture8

Modification of gene expression
Genetic strategies
To take into consideration:
Means:
9/11-2010
- transcription
- promoters
- translation
- terminators
- localization
- RBS strength
- number of gene copies
- protein stability
- gene fusions
- metabolic conditions
- gene sequence (codon usage) - purification process etc
- etc
Promoter strength affects the protein expression level
Promoter (Escherichia coli)
Recombinant protein production:
Prokaryotes II
Start of
RNA
synthesis
-35
Start of
coding
sequence
-10
CCGGTTGACAGATAGTCGTGTATGCGATATAATCAGCCCGTAGTCGGAGGGTCCTGACATG…
GGCCAACTGTCTATCAGCACATACGCTATATTAGTCGGGCATCAGCCTCCCAGGACTGTAC…
"Pribnow box"
P
Gene for protein
5´
Produced mRNA molecule
Translation
Figure 6-3 Molecular Biology of the Cell (© Garland Science 2008)
Commonly used promoters
Promoter
Inductiona
Strength
lac
tac
-35 trp -10 lac
trc
trp
T7
PL(λ)
lac(TS)
PSPA
PBAD
IPTG
IPTG
IPTG
Trp-depletion/β-IAA
IPTG
Heat
Heat
Constitutive
L-arabinose
rel. weak
rel. strong
rel. strong
Test of promoter strength
His6ABPGFP
Trc
very strong
very strong
T7
LacUV5
SPA
Negative
control
weak
rel. strong
amost
frequently used means of induction.
Abbrev.: SPA, Staphylococcus aureus protein A;
IPTG, isopropyl-β-D-thiogalactopyranoside; β-IAA, β-indoleacrylic acid.
Fluorescence intensity
Red = T7
Green = Trc
Blue = LacUV5
Yellow = SPA
H. Tegel, unpublished
1
CAP
-10
Lac I
S.D.
5´
mRNA-start
RNA-pol.
-35
DNA
-10
cAMP/CAPbinding
region
Operatorsequence
S.D.
5´
3´
Lac-repressor
(Lac I)
Lactose or
IPTG
(synthetic analogue)
Lac Z
Lac I
mRNA
Lac I
Promoter=
”Landing spot” for
RNA polymerase
repressor
Catabolite
Activator
Protein
(CAP)
Promoter
-35
Cyclic
AMP
(cAMP)
Lac Z
Lac Y
S.D.
Lac Y
Lac A
S.D.
mRNA
Lac A
3´
promoter
Trp
Trp-operon
Regulation of the lactose operon (E. coli)
operator
trpL
trpE trpD trpC trpB trpA
β-galactosidase:
Lactose permease: Thio-galactoside-acetylase:
Turns lactose into Regulates the lactose Degrades non-cleavable
galactose and glucose
uptake
lactose analogues
In the absence of lactose:
• Lac-repressor binds to the operator sequence
• Transcriptionen is blocked; no β-galactosidase (or any of the other enzymers) is producced
Tryptophan depletion
repressor
Lactose (or IPTG) present:
• Lac-repressor cannot bind to the operator sequence
• Transcription is allowed; β-galactosidase (and all the other enzymes) is produced
trpL
Presence of lactose (or IPTG) and low concentrations of glucose:
• cAMP concentration rises
• cAMP-CAP-complex formed that can bind upstream of the promoter
• cAMP-CAP-complex promotes transcription (“guides” the RNA polymerase)
• More β-galactosidase is produced
trpE trpD trpC trpB trpA
mRNA
T7-system & PL
pET protein expression system
T7-system
Lac-promoter
LacT7 RNAoperator polymerase
gene
T7-promoter
gene of interest
mRNA
mRNA
T7 RNA-polymerase
PL-promoter
Repressor protein from bacteriophage λ
Growth at 28-30°C:
active repressor
Induction at 42°C :
repressor is inactivated, result in transcription
PBAD promoter
FIGURE 10.4
Replicons (ori) carried by plasmid vectors
Plasmid
Replicon
Copy nr.
References
pBR322
pUC
pMOB45
pACYC
pSC101
colE1
pMB1
pMB1 deriv.
pKN402
p15A
pSC101
colE1
15-20
500-700
15-118
18-22
~5
15-20
Bolivar et al. (1977)
Viera & Messing (1982, 1987)
Bittner & Vapnek (1981)
Chang & Cohen (1978)
Stoker et al. (1982)
Kahn et al. (1979)
Incompatibility groups:
colE1, pMB1
IncFII, pT181
P1, F, R6K, pSC101, p15A
FIGURE 10.5
2
Plasmids - a burden for the host cell
• Plasmids are lost
• Strategy: plasmid encoded protein that is crucial
for survival in the cell culture
• Usually antibiotics or essential metabolite has to
be added (expensive!)
• Risk of gene transfer (e.g., MRSA)
• Solution: Integration of the DNA on chromosome
Expression optimization: translational level
Expression optimization:transcriptional level
• Promoter strength:
- degree of ”consensus” sequence
• Sigma factor availability:
- growth condition dependent
• Positive/negative regulation
• Enhancer/silencer regions
Heterologous protein expression in E. coli
• Common problems • Solutions
- protein aggregation
- decreased synthesis rate
• RBS-sequence dependent
- protein misfolding
- inactive protein
- low yield
UAAGGAGG mRNA
AUUCCUCC 16S-RNA
• Distance: RBS-ATG
• Secondary structure of the mRNA
• ”Codon usage”
Expression of a eukaryotic gene in bacteria
(weaker promoter, reduce conc. of inducer, low
temperature)
- co-expression of folding
modulators
- fusion tags
- protein engineering of target
- host engineering
Codon usage: E. coli
FIGURE 10.1
3
Codon usage: H. sapiens
Codon usage affects the translation rate
FIGURE 10.3
Problems due to rare codons
• reduced translational rate
• low expression levels
• amino acid misincorporations
Solutions to problems caused by rare codons
• exchange the rare codon/s for a more
frequently used codon
• introduce extra copies of the “limiting” tRNA
genes
• truncated or amino acid-deleted proteins
• frame-shifted proteins
Chaperone-assisted protein folding (cytoplasm)
Role of DnaK and GroE chaperone machines in nascent
protein folding
Georgopoulos, C. Genetics 2006;174:1699-1707
Baneyx F et al, Nature Biotechnology (2004), 22: 1399-1407
Copyright © 2007 by the Genetics Society of America
4
Effect of co-expression of GroEL/ES
Cytoplasmic chaperones
Family
Name
Hsp100
Clp
Cofactors
Function
Substrate specificity
Disaggregase
Regions rich in aromatic and
bacic aa
+
Hsp90
HtpG
Hsp70
DnaK
DnaJ, GrpE
Folding/secretory
chap.?
Unknown
+
Folding chaperone
Segm. of four to five
hydrophobic aa, enriched in
leucine and flanked by basic
residues
+
Hsp60
GroEL
GroES
Folding chap.
α/β folds enriched in
hudrophobic and basic
residues
+
Hp33
DJ-1
superfam.
Hsp33
Holding chap.
Unknown
-
Hsp31
Holding chap.
Unknown
-
Small
Hsps
IbpA, IbpB
Holding chap.
Unknown
-
PPIase
TF
Hold. chap., PPIase
Eight aa motif enriched in
aromatic and basic residues
-
SecB
SecB
Secretory chap.
Nine aa motif enriched in
aromatic and basic residues
-
P
ATP
requirement
HSD1
EGFP
time: 4 h
DH5alpha/11b-HSD1-eGFP
DH5alpha/11b-HSD1-eGFP/GroELES
Export and periplasmic folding pathways
Periplasmic chaperones
Classification
Protein
Substrates
Generic chaperones
Skp (OmpH)
FkpA
Outer membr. proteins and misfolded periplasmic
proteins
Broad substrate range
Specialized chap.
SurA
LolA
PapD
FimC
Outer membrane proteins
Outer membrane lipoproteins
Proteins involved in P Pili biosynthesis
Proteins involved in type 1 pili biosynthesis
PPIases
SurA
PpiD
FkpA
PpiA
Outer membrane beta-barrel proteins
Outer membrane beta-barrel proteins
Broad substrate range
Unknown
Proteins involved in
disulfide bond formation
DsbA
DsbB
DsbC
DsbG
DsbD
DsbE (CcmG)
CcmH
Reduces cell-envelope proteins
Reduces DsbA
Proteins with nonnative disulfides
Proteins with nonnative disulfides
Oxidised DsbC, DsbG and CcmG
Cytochrome c biogenesis
Cytochrome c biogenesis
Baneyx F et al, Nature Biotechnology (2004), 22: 1399-1407
E. coli: periplasm
Folding in the bacterial periplasm: protein disulphide-isomerases
• DsbA, a generic dithiol oxidase in the periplasm of E. coli
E. coli: periplasm
Folding in the bacterial periplasm: protein disulphide-isomerases
•
DsbC catalyzes disulphide bond exchange reactions
5
E. coli: periplasm
Known components of the thioredoxin system and
glutaredoxin system.
Folding in the bacterial periplasm: peptidyl-prolyl cis/trans-isomerases
•
•
•
polypeptide bonds are synthesized in trans configuration
5% cis peptidyl-prolyl bonds in native proteins
Ea = 20 kcal/mol for peptidylprolyl-isomerisation
 Rate-determining step of protein folding
Prinz W A et al. J. Biol. Chem. 1997;272:15661-15667
©1997 by American Society for Biochemistry and Molecular Biology
trxB/gor mutants allow disulfide formation in the
cytoplasm
Prinz W A et al. J. Biol. Chem. 1997;272:15661-15667
©1997 by American Society for Biochemistry and Molecular Biology
Urokinase becomes active in the cytoplasm of WP759
and WP778
Prinz W A et al. J. Biol. Chem. 1997;272:15661-15667
©1997 by American Society for Biochemistry and Molecular Biology
Low temperature adaptation
Metabolic load
• Plasmids with high copy-number
• Oxygen limitations
•Overexpression of foreign proteins may lead to
shortage of tRNAs
• Export machinery can become blocked. The export
of endogenous host cell proteins is then obstructed.
• Metabolic properties way be changed in a negative
way.
• Foreign proteins can be detrimental to the host cell,
e.g. toxic properties
Ferrer M et al, Nature Biotechnology (2003) 21: 1266-67
6
Oxygen limitations
•
Decreased growth rate, changed
metabolism
•
Stress response is activated;
protease expression is upregulated
Solutions:
•
•
Protease negative strains will result in
less-pronounced protein degradation.
However, faulty (mis-folded, truncated etc)
proteins cannot be degraded.
What happens to the cell?
• Cell growth decreases, low cell densities
• Shape and size changes
• Stress response. Proteases are expressed
• Reduced stringency in DNA-proof reading
• Wrong amino acid can be incorporated due to
insufficient amounts of tRNA or amino acids.
Expression of a bacterial
haemoglobin can improve
intracellular oxygen binding.
How can we reduce the metabolic load?
• Use other codons (synonymous)
• Use tightly regulated promoters (they should not
”leak”)
• Reduced expression level may result in higher
cell density in the culture, and thereby better
productivity.
7