it was first identified (1885) by theodor escherich as a
Post on 13-Jun-2022
4 Views
Preview:
TRANSCRIPT
THE MOST POPULAR AMONG BACTERIAL HOSTS IS
It was first identified (1885) by Theodor Escherich
And it is, now, the world-wide most employed microorganism in both research and applied labs
as a commensal of human gut
ESCHERICHIA COLI
Every single gene of the E.coli K12 type strain (MG1655)
Most of the knowledge on transcription, translation, DNA replication, genetic code, trasducing phages and so on, have been acquired by studying E. coli
Has been silenced
to determine which genes are
essential
and which are not
It is a Gram-negative bacterium belonging to
γ-proteobacteria Proteobacteria Enterobacteriaceae
didermal structure
•CELL WALL
PERIPLASM
•CELLULAR (inner) MEMBRANE (IM)
•cytoplasm
•peptidoglycan
•Outer membrane (OM)
PERIPLASM an oxidizing environment, ~ 4% total proteins
CYTOPLASM a reducing compartment
OUTER MEMBRANE Where surface epitopes can be expressed
Straight rod ~ 2 µm x 0,5 µm
4 main phylogenetic groups (A, B1, B2, D)
Plenty of serovars
O (>170) = polysaccharide
chains LPS
H (hauch, german for «mist, whiff») (>50) = flagellar proteins
K (Kapsel, german for capsule) (>100) = capsular antigenes (microcapsular
equivalent)
Antigens: O: … H… K…
Escherichia coli K-12
Model microorganism
A very useful biotechnological tool
K12
RESPIRATION
AEROBIC OR
FERMENTATION
IT GROWS WELL ON RICH MEDIA (37 -42 °C) BUT CAN GROW IN THE RANGE 10-45 °C
ANAEROBIC
WITH A MEAN GENERATION TIME = 20’
ATP (ENERGY) IS PRODUCED BY
IT IS ABLE TO ADAPT TO SEVERAL CONDITIONS
E. coli is the most efficient and widely-used host for recombinant protein production and for the production of DNA plasmid to be used to transform other hosts
Well-known genetics
Plenty of mutants are available
Well-known fermentations pathways
Make it easier to set and control industrial processes
Although not naturally competent, it can reach a high transformation efficiency
Fast growth in many media
E. coli provides large biomasses, and its cells are very easy to lyse
Pathogenicity class = 1 Safe to be
used
The most common origin in the plasmid vectors employed for E. coli, is the ColE1/pMB1 one
ColE1 = pMB1: 1 base
ColE1/pMB1 harbouring plasmids have a relaxed control
By halting the protein synthesis, the plasmid still replicates, as the polymerases are very stable enzymes. So, the ratio plasmid vs cellular debris can be modified
The ColE1 family encompasses multicopy plasmids in the range 15-
20 ~ 500 copies/cell
A LOT OF PLASMIDS VECTORS ARE AVAILABLE FOR E. COLI
P15 (from a natural E. coli plasmid) Relaxed replicon, 20-40 copies
The plasmids harbouring this replication origin can coexist with ColE1/pMB1 in the same cell
Further replicons (different incompatibility groups)
pSC101 (from a natural Salmonella plasmid)
Stringent Replicon, very low copy number (1-5)
F (natural plasmid Fertility factor) and φ (bacteriophage) P1 origins
1-2 copy/cell, used for artificial chromosomes (BACs & PACs)
Compatible with both p15 e ColE1/pMB1
e.g. pACYC184
conditional replication systems (suicide vectors)
e.g. The R6K replication occurs at three sites on the plasmid called the alpha, gamma and beta origins
In E. coli the pi protein can be supplied in trans by a prophage (lambda pir) that carries a cloned copy of the pir gene: the plasmid will replicate in a “lambda pir” host, but will unable to do so in other E. coli strains
It requires the “pi protein” encoded by the pir gene, to function
Plasmids endowed of the oriR6K will not be able to replicate in the absence of the pir gene product
Conditional (temperature sensitive) ORIs are also largely employed: they are based on mutated replication enzymes which are inactivated at >30 °C
conditional replication systems are usually employed in order to obtain chromosomic integrations of heterologous fragment, or alleles exchange
SELECTION MARKERS
Escherichia coli is sensitive to
In bacteria, antibiotics are used almost exclusively
KANAMYCIN
CHLORAMPHENICOL
TETRACYCLINE
AMPICILLIN
only few cells take up DNA We need selectable markers to detect them
A selective agent kills or prevents the growth of those cells that do not contain the foreign DNA, leaving only the desired ones
Is a β-lactam antibiotic, which interfers with the peptidoglycan synthesis
AMPICILLIN Its target is the transpeptidation reaction: PBPs act by removing the terminal D-Ala residue and creating
the trans-peptide bond
β-lactam antibiotics act as pseudosubstrates for the Penicillin Binding Proteins by stably
acylating their active site
Serine active site
D-A
DAP
D-A
D-A
DAP
D-A
R H CH3
N
S
CH3 COO- O
β-lactam
BLA_TEM
Penicilloic acid (inactive)
HN
R H CH3 S CH3 COO-
O O
TPase TGase TPase TGase
Which acts mainly on penicillins, by hydrolizing the β-lactam ring, and preventing them to bind to PBPs
The resistance determinant used on plasmids (blaTEM) encodes a periplasmic class A β-lactamase
PBPs
Easy to use Low costs Fast growth of the resistant colonies
AMPICILLIN
The Ampr cells secrete the enzyme
Excellent choice for the laboratory
DRAWBACKS, DISCOURAGING FOR THE LARGE PRODUCTIONS
R
plasmidless cells can grow S S
The antibiotic concentration decreases
ADVANTAGES
On solid media, this is evident by the appearing of satellite colonies
A possible alternative is the carbenicillin, more expensive but stabler
In liquid media the loss of plasmid is high (up to 80%)
S S
S
S S
S
S S
S
R
-Ampicillin degrades spontaneously at acidic pH
If the selective agent is ampicillin, the starter cultures should not grow more than 8-10 hs
-if not correctly stored, the activity decreases quickly
CHLORAMPHENICOL
It blocks protein synthesis by binding to the 50s ribosomal subunit
The determinant used as a marker (Chloramphenicol Acetyl Transferase - CAT) derives from the TN9 transposon
CAT is a cytosolic, tetrameric protein
Cat + CM + acetyl CoA hydroxy-methyl derivatives, unable to bind to the target
DRAWBACKS: Chloramphenicol slows the bacterial growth rate
it is toxic and potentially carcinogenic
It is not allowed for productions aimed to human use
It has to be dissoved in ethanol and a particular attention must be paid in mixing the antibiotic to the medium as microclumps could occurr
TETRACYCLINES
H H
H O
H
CONH2
H O H O
H O H O
N(CH3)2 CH3
O O
Are a large group of antibiotics with a molecular structure containing four rings
They are naturally produced, by Streptomyces aureofaciens or S. rimosus, or semisynthetic
Tetracyclines act by forming a stable bond with the 30S ribosomal subunit, so deforming the "A“ site
for the selection just a small amount of the hydrochloride (usually 5 μg/ml) will be sufficient
HCl
The gene used for the selection in E. coli vectors is tetC from pSC101, which encodes a tetracycline efflux system, regulated by the repressor TetR
DRAWBACKS: a limited solubility (0.4 mg / mL in water; 20 mg / mL in alcohol) and photolability
P A E
30S
P A E
30S
AA2
TETRACYCLINES
H2SO4
KANAMYCIN
produced by Streptomyces kanamyceticus, it is the most frequently employed among amynoglicosides
the genic determinant used for plasmid selection is derived by the Tn 903 transposon and confers resistence to both kanamycin and neomycin
It acts by interacting with at least three ribosomal proteins, so inhibiting protein synthesis and increasing translation errors
The product is APH(3')I, an Aminoglycoside 3'-phosphotransferase
For the selection, kanamycin sulfate is used, generally at a 30-50 μg/mL concentration
AMP TET
TET
AMP
Antibiotic resistance genes can be used also for the SCREENING, (older plasmids) but this implies a longer time for handling
E.g. cloning within the tet gene, that is therefore disrupted
Day 1: after having selected on AMP, the transformant colonies are streaked or replicated on tetracycline plates
Day 2 : the clones unable to grow on tetracycline, harbour a charged plasmid and must be picked from the first plate
AMP
The best suited screening markers are those conferring features that can be detected by hystochemical methods
α-peptide of the β-galactosidase (blue/white)
It requires a defective host strain, deleted in the α-peptide encoding region so to be complemented by the plasmid
Reagent: X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside)
The insertional inactivation is obtained by cloning the DNA fragment within the marker encoding gene
Some other selection systems can be used with every E. coli strain (i.e. they do not depend upon mutations in the host genome)
phoC (Morganella morganii): aspecific phosphatase, can be detected by adding BCIP (5-bromo-4-Chloro-
3 indolyl-phosphate) to the agar medium
Melanine-like pigment of Streptomyces: can be detected by adding tyrosine to the agar medium
The insertional inactivation is obtained by cloning the DNA fragment between the marker encoding gene and the promoter
It has, however, a rather low sensitivity and the browning is not clear cut on the isolated colonies
PROMOTERS
A good promoter must:
be strong enough
to function in many (most of) E. coli strains
inexpensive
simple
Independent from the normal components of culture media
To be induced in a manner
T78T82G68A58C52A54 -- 162117521819 -- T82A89T52A59A49T89 -35 spacer Pribnow (-10)
That is to be as much as possible consistent with the E. coli consensus
a strict control allows to obtain a good biomass yield before starting to express the product
A tight control is needed because
The presence of heterologous proteins can induce/activate the host proteases
The product could be toxic to the cell
Or could aspecifically interact with some cellular components
By binding or damaging DNA
By sequestering essential proteins
Through an improper interaction By causing oxydative
stresses or inducing response systems
“UP” SEQUENCES are «optional» promoter features
A+T RICH SEQUENCES LOCATED UPSTREAM THE -35
By binding to the alpha subunit of the RNA polymerase
UP sequences have been observed in
They are less conserved than -35 and -10 regions Their consensus is [-59 nnAAA(A/T)(A/T)T(A/T)TTTTnnAAAAnnn -38]
Whenever present, they increase the expression level (30-70%)
Monoderms (Bacillus, Clostridium)
Didermes (E. coli)
Bacteriophage (λ and Mu)
Shine Dalgarno start of the ORF A good SD region, in E. coli is about 6-8-bp long
consensus 5’-UAAGGAGG-3’
The start codon is usually AUG Some microbial species with an high G+C can use alternative codons ( the most frequent one is GUG) To express these ORFs in E. coli, it is expedient to modify the start codon
The bp number
4-14 possible
7-9 Most frequent
8 OPTIMAL
UAAGGAGG-xxxxxxxx-AUG
The quality of the bases A+T OK
G+C NO!
C and G negatively bias the translation efficiency
In the region spacing from SD and the start codon, it is very important to check:
The best thing would be to calculate the translation rate for each individual CDS, in terms of thermodynamics
It’s also necessary to avoid palindromic sequences involving the ORF start or masking the SD region
ccugaauUAAGGAGGnnnnnnAUGauucagg
UAAGGAGGacucgagaAUGnnnnnnnnucucgagu
And, if needed, to modify the SD sequence according to it (the translation rate should not exceed the folding and secretion ones)
This is almost impossible to do experimentally, but there are programs that can help, such as
the RBS Calculator at http://salis.psu.edu/software
Induction
The growth does not change after the induction
The growth rate decreases
The effects of the overexpression are depicted by the growth curve of the host
check for lysis
If the host cells lyse, consider the lysis amount, the related loss of product
decide whether to try other conditions and/or induction times, or not
Favourable curves
Induction
Costitutive Espression of a toxic product
The growth is inhibited/ the host cells lyse
Negative curves
Promoters frequently used in E. coli
lac promoter
INDUCTION: ALLOLACTOSE (lactose byproduct)/IPTG YIELD: MEDIUM to LOW REGULATION: NEGATIVE (Repressor: LacI) CONTROL: INADEQUATE
Z Y A O P I repressor β-galactosidase
permease promoter
operator
LacZ + HOH
LacZ + HOH
glucose
galactose lactose
allolactose
Due to the basal expression, a few molecules of LacZ and LacY are there
when lactose is made available
so to convert lactose to allolactose and relieve the repression (induction)
Z
Y
negative regulated genes are expressed at low (basal) level in the bacterial cell
Z
Y
Plac is a rather leaky promoter: its basal expression is hardly limited
Unless the repressor is located on the same plasmid
In the lab, induction is often not actually necessary
or the host is a LacI overespriming (LacIq) one
IPTG is a gratuitous inducer
it binds to the repressor that is no more able to bind the operator
but it is not metabolized
the concentration remains constant and can be controlled
However, it is relatively expensive (lactose can be used instead)
Z Y A O P I
IPTG is not allowed in GMP for human use, further limiting the usefulness of pLac for
industrial productions
TTTACA TATGTT TGGAATTGTGAGCGGATAACAATT Plac
it differs from the consensus in 3 nucleotides
the spacer length is sub-optimal (18 nt)
the spacer length is still non ideal
TTTACA TATAAT TGGAATTGTGAGCGGATAACAATT lacUV5
Although largely used for in the lab applications, Plac is not a strong promoter
The derivate PlacUV5 is much stronger, due to a 2 bp mutation in the -10 hexamer, that enhances the recruitment of the RNA polymerase σ70 subunit
WILD TGTGAGTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCT UV5 ...A...................................................
-65 -35
-10 +1 Operator RBS WILD CGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATG UV5 .....AA................................................
The net effect of the three-point mutations is the creation of a stronger promoter that is less sensitive to the glucose effect
Moreover, a third point mutation located in the CAP/cAMP binding site, decreases the affinity of PlacUV5 for CAP/cAMP and the sensitivity to
catabolite repression
The good recruitment of σ70 bypasses the need for a positive activation (CAP) typical of the wild-type Plac
PtacI
Ptac ensures a good yield: higher than Plac but not so high as the T7 promoter
INDUCTION: IPTG YIELD: MEDIUM-HIGH (5x than the parental ones) REGULATION: LAC I BASAL EXPRESSION : HIGH
-35 -10
lac operator
TTAACT TTGACA trp
TTTACA TATAAT AATTGTGAGCGGATAACAATT lacUV5
TATAAT AATTGTGAGCGGATAACAATT TTGACA RBS tac I
Its strenght could be a drawback for toxic products and membrane proteins
It is a synthetic hybrid promoter derived from the E. coli trp and lacUV5 promoters
tetA
INDUCTION: ANHYDROTETRACYCLINE
YIELD: MEDIUM TO HIGH
REGULATION: NEGATIVE
CONTROL: TIGHT
BASAL EXPRESSION: LOW
P tetA
TetR heterologous gene
-
The repressor encoding gene is placed on the plasmid: that’s why the system does not depend from the background of the host strain
NOT DEPENDENT UPON THE STRAIN AND/OR THE METABOLIC STATE OF THE HOST THE INDUCER MOLECULE IS NOT EXPENSIVE
The induction is performed with ANHYDROTETRACYCLINE at low concentrations
A full induction(~50 ng/ml) does not bias at all the growth rate of E. coli
OH
OH OH
OH
CH3 N(CH3)2
O
O CONH2 O
A-TET TET Binding efficiency
35
1
A-TET TET Bactericidal action
100
1
This promoter has been successfully used to produce many heterologous proteins (FABs toxins..)
Non-induced cells induced cells
1 100 =
INDUCTION: ARABINOSE DOSE DEPENDENT
YIELD: MODULABILE
CONTROL: POSITIVE
REGULATION: TIGHT
BASAL EXPRESSION : VERY LOW
AraBAD The promoter of this tightly
controlled operon, is a suitable tool for the heterologous
expression in E. coli
CTGACG -- 18 -- TACTGT TTGACA -- 17 –- TATAAT
araBAD
consensus
Inducer=Arabinose: very cheap and suitable for the Good Manifacturing Practices
The almost totally lack of a basal expression, in arabinose free media added with glucose, balances the structural weakness of this promotor
P AraC + heterologous gene
0.001%
ARA
3
1%
ARA +
The arabinose amount needed to induce expression depends upon the genetic background of the host strain
rhaPBAD
RhaR transcriptional activator 1 RhaS positively regulates the entire regulon (rhaBAD: catabolism and rhaT: transport)
INDUCTION: RHAMNOSE
YIELD: MEDIUM
REGULATION: TIGHT AND ELABORATE
CONTROL: TIGHT
BASAL ESPRESSION: LOW
RhaR
RhaS
L-rhamnose
RhaB RhaA RhaD
RhaT The regulon is however also subjected
to a catabolite repression
The optimal time to induce and to collect the product must be determined experimentally and carefully
the choice between lysogenic and lytic cycle depends upon the balance between CI and CRO The CI product is the
Lambda repressor
If CI is already expressed in a bacterial cell (that is: if there is a
prophage)
No other lambda phages are able to infect the same cell
att
cII
Q P
O
R S
Cos
cI
A W B
C D
E F Z
U V G T H M L K I J
cro PR PL N cIII
int
xis
λphages can enter a lytic (right arm) or a lysogenic (left arm) cycle
Phage promoters (Lambda and T7)
Bacteriophages offer a possible alternative to the regulated promoters of metabolic genes
cII
Q P O
R S
cI
A W B C
D E F Z U V G T H M L K I J
cro PR PL N cIII
int
xis
PL and PR promoter are directly recognized by the bacterial RNA
polymerases
And are very efficiently regulated by the Lambda
repressor
The Lambda PL/OL promoter-operator ensures medium to high expression levels
λPL
To further improve the control on the promoter, mutated forms of the CI repressor are usually employed
to easily switch the expression on/off, a temperature-sensitive mutant of the lambda repressor (cI857; CT in 37742) is the most frequently used
29-30 °C λPL
T sensitive cI857 repressor: ON
Active form
Heterologous gene
λPL = repressed
42 °C λPL
T sensitive cI857 repressor: OFF
Inactive form
Heterologous gene
λPL = constitutive
It is very advantageous for the overexpression of proteins susceptible to proteolysis which would be degraded at more growth-friendly temperatures
An alternative to the use of temperature sensitive mutant is to put the CI repressor under the control of PTrp
An economic and practically devoid of tryptophan culture medium contains molasses and acid casein hydrolysate
Heterologous gene
P trp cI repressor λPL
By adding tryptone (tryptic digest of casein tryptophan rich) to the medium)
Heterologous gene λPL cI repressor P
trp the repressor
expression halts the foreign gene expression starts
Another interesting application of CI repressor has been proposed in 2016 by G. Durante- Rodríguez et al.
These authors engineered a artificial chimeric regulator
By fusing the DNA binding domain of CI and the BzdR repressor of the bacterium Azoarcus, that responds to benzoyl-CoA
In natural conditions the lytic cycle is triggered by the SOS response, trough the action of RecA
PR promoter: repressed PR promoter: derepressed
λNTCI λCCI stress
Benzoyl-CoA
PR promoter
λNTCI
LCBzdR
PR promoter: derepressed
Differently from the Lambda PL one, the T7 promoter is not recognised by E. coli RNA polymerase as it promotes the class 2 (tardive) genes
So while choosing to use a T7 promoter we need a bacterial host expressing T7 RNA polymerase
The transcription from the T7 promoter depends upon the T7 RNA polymerase, produced among the class1 (early) genes which are transcribed
by the host RNA polymerase
the T7 RNA polymerase is faster than the E.coli one and its expression has to be tightly controlled
BL21 DE3
The most popular one is the commercially available E. coli BL21 (DE3)
A lisogenic E. coli «B» strain harbouring the DE3 Lambda phage
In the λDE3 phage the T7 polymerase is expressed from the LacUV5 promoter so that it can be succesfully produced even in
the presence of glucose
The λDE3 mutant has been obtained by inserting the PlacUV5-T7 fragment within
the integrase encoding gene
The disruption of the int gene prevents the excision of the
DE3 prophage
Of course, it is possible to construct one’s own lysogen (DE3) strain, with the same technique used to construct the BL21(DE3)
or other commercially available (DE3) strains
To do so 3 (4) different bacteriophages are needed:
λDE3 (int-): it is not able to integrate into (or excise from) the bacterial chromosome by itself
An Helper Phage (B10) that provides the int function to λDE3, but cannot form a lysogen by itself because it is cI- (has no repressor)
By means of the integrase provided in trans by the Helper Phage, the DE3 will be able to integrate in some bacterial cell
However, the colonies of the lysogenic cells would grow among an overwhelming number of WT ones, so we need a selection tool
Both of these phages cannot propagate due to another mutation affecting the ability to lyse the bacteria
-Cannot kill λDE3 lysogens, because it has the same immunity, as λDE3 -Cannot integrate in susceptible cells (cI-) -It kills the mutated bacterial cells resistant to λDE3 that otherwise would survive and hamper the screening
The selection phage (B482)
λDE3
B10 B482
Lysogens are prepared by co-infection with the three phages
B10 + λDE3 integration
Unaffected by B482
No DE3 integration
Killed by B482
most of the growing colonies should be (DE3)
To check the lysogenic state a 4th phage is employed
But it will succeed in killing the λDE3 lysogen cells, as they produce T7
polymerase, once induced with IPTG
The Tester Phage, is a T7 mutant, deleted in the T7 RNA
polymerase region
So it is unable to form large plaques on WT E. coli cells
A further control may be exerted with the T7 lysozyme
The LSZM encoding gene has been cloned in a p15 plasmid obtaining pLysE and pLysS
Lysozyme binds directly to the T7 RNA polymerase hampering its activity
But it has the drawback to slow the host growth rate as it cuts the cell wall, weakening the cell structure
p15 Cmr
pLysE
T7 LSZM
p15 Cmr
pLysS
T7 LSZM
Notwithstanding the tight control, some basal activity still occurs; it is not usually a problem but it could be if the product is toxic
pLysE and pLysS differ only for the orientation of the T7 lysozyme encoding gene
But it is a very important difference
In pLysE the gene is transcribed from the very strong Tet promoter
p15 Cmr
Prom Tet
T7 LSZM
pLysE However, the amount of produced lysozyme is too high
The cells became too frail
Actually, this was the projected construct
As the gene was transcribed from the weak T7Φ 3.80 promoter
p15 Cmr
T7 LSZM
pLysS
prom T7Φ 3.80
T7Φ 3.80 is located DOWNSTREAM of the gene but transcribes it together with the
entire plasmid
In this case the produced lysozyme is sufficient to hamper the T7
polymerase activity
without halting the growth of the culture
unexpectedly, the opposite orientation seemed to function
A T7 driven expression can sometimes cause some aggregation of the produced proteins
A possible solution is
To slow the production rate
By dosing the IPTG (25-100 µM instead of 1 mM)
By decreasing the incubation temperature
INDUCTION
After the induction, the product output steps up abruptly but
the growth of the hosts harbouring a T7 driven plasmid could stop
it is essential to carefully determine the right timing in order to get the maximal yield
T7 RNA polymerase
CE6 BACTERIOPHAGE
The polymerase gene is cloned into the int gene so to be transcribed from the λPL e λPI promoters during the infection
λCE6 is not able to enter the lytic cycle because of the “Sam” mutation (A G in 45352) which inactivates the lysis protein GPS
the basal expression of T7 RNA polymerase prevents the use of lysogenic hosts with particularly toxic genes transcribed from the T7 promoter
CE6
Immunity region
cII
Q P
O
cI857 cro PR PL N
cIII
xis
PI
int
Under the control of CI857
In such cases the λCE6 bacteriophage can be used to provide a source of T7 RNA polymerase to susceptible hosts
When CE6 infects the cell the T7 RNA polymerase synthesized de novo starts to copy the target DNA with a very high efficiency
CE6 can be propagated in the E. coli LE392 (supF) strain which suppresses the Sam7 mutation allowing the phage
to enter in the lytic cycle
the system is not so efficient as the use of lysogenic strains (DE3)
But until the infection is performed there are no polymerases able to recognize the T7 promoter
and to transcribe the target gene! +T7 POL
Another possible drawback of the T7 promoter in lysogenic strains is is the uneven expression in the single transformant clones
BL21 DE3
BL21 DE3
BL21 DE3
BL21 DE3
BL21 DE3
so that it is necessary to examine several colonies to look for those with the highest production level
T7/lac can also be combined to the use of pLysS
Another variant is the T7/lac hybrid promoter
P T7
lac operator Heterologous gene +1
The basal transcription is blocked by LacI
relieves at the same time the repression from the target gene Very often the lacI gene is included in the vectors with this system, to warrant a tight control
P T7
lac operator Heterologous gene +1
The IPTG used to induce the T7 RNA polymerase expression in BL21(DE3)
LacI
LacI IPTG
Another important feature is the TERMINATOR
On the expression vectors, a strong rho independent terminator is often located downstream from the MCS to ensure the right release of the mRNA
from the ribosome
Terminators are palindromic sequences followed by a A strand (U at the 3’ end of the mRNA)
<<<<<<<:::<:<<:-:--:-:>>:>:::>>>>>>> AACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG T7
<<<<<<<<<<<<<<<<<<---->>>>>>>>>>>>>>>>>> AAAACAAAAGGCTCAGTCGGAAGACTGGGCCTTTTGTTTT
rrnD
The terminator prevents the genes located downstream the target one, and in the same orientation, to be co-transcribed from the promoter
Downstream gene P Target gene
Downstream gene P Target gene
On some plasmids for example the β-lactamase encoding gene can be transcribed from the T7 promoter
BLA
The degradation rate of ampicillin increases
S S
Plasmidless cells can overgrow
If there are other promoters even far from the target ORF but oriented in the same direction
So to o prevent undue transcripts involving the gene of interest
It may be expedient to put a strong terminator also UPSTREAM of the expression cassette
WHERE IS BETTER TO DIRECT THE RECOMBINANT PRODUCT IN THE CELL?
Inclusion bodies easy purification protection from proteases inactive (non toxic) proteins Higher yield Simpler plasmid design
Simple purification low proteolysis level Improved folding N-terminus authenticity
The least extensive proteolysis Simple purification Improved folding N-terminal authenticity
Usually no secretion Cell lysis
Signal does not always facilitate the export Inclusion bodies may form
Inclusion bodies protein folding denaturation/refolding N-terminal extension
cytoplasm
periplasm
Outside the cell
The macromolecule concentration can reach 300–400 mg/ml
E. coli cytoplasm is a very crowded environment
On average, a ribosome releases one protein chain every 35 seconds
In such conditions the first challenge for a protein is to correctly fold
The small (<100 residues) single domain host proteins efficiently reach a native conformation owing to their fast folding kinetics
CYTOPLASM
large multidomain and overexpressed recombinant proteins often require the assistance of folding modulators
IN VIVO THE PHYSIOLOGICAL PROTEIN FOLDING IS ASSISTED BY CHAPERONES
the nascent polypeptides first interact with ribosome-bound Trigger Factor (TF)
TF transiently associates to the ribosome
To form a protected folding space where nascent polypeptides are shielded from both proteases and aggregation
Once released from TF, the peptides can either
fold spontaneously (roughly 70% of cytosolic proteins under normal growth
conditions)
Or require further folding-assistance by downstream
chaperones
KJE system (5-18%) DnaK (Hsp70) with its co-chaperone DnaJ and the
nucleotide exchange factor GrpE
ELS system 10-15% GroEL (Hsp60) and its co-chaperone GroES
The substrate proteins ejected from DnaK can
DNAK & DNAJ stabilize the nascent chain in a folding-competent state already during translation
By binding hydrophobic segments that are exposed in the extended chain but that will be later buried within the
folded protein
K
Be transferred in a semi-folded form to
GroELGroES
fold into a proper conformation
Be recaptured by DnaKDnaJ for additional cycles of binding/release
Proteins requiring a folding assistance can enter
GroEL is made of two rings and the protein binds inside the open one
the open ring becomes an enlarged folding chamber with a hydrophilic lining, where the substrate folding (~10s) is timed by
ATP hydrolysis
the GroEL ring binds ATP
And rapidly recruits GroES, which caps the cavity
EL EL EL
EL
EL EL
ES
ES
Once folded, the peptide will be released when ATP will bind again and a new peptide
will enter on the opposite side
In the meanwhile, GroES, the bound ADP and the folded peptide from the previous cycle, are ejected from the opposite ring
If the ejected substrate still exhibits significant surface hydrophobicity it will be recaptured, entering a new cycle
ES
ES
EL
EL
The chance to succeed, however, critically depends on the structure of the heterologous
protein
Direct the peptide to the PERIPLASM
To facilitate the correct folding of recombinant proteins
Some vectors include the DnaK/DnaJ or GroEL/GroES encoding genes
As the cytoplasm is a REDUCING compart, proteins that depend upon the disulphide bond formation, are easily misfolded
OR
In such cases it is often more expedient to manipulate the host strain rather than rely on plasmid features
In eukaryotic cells, disulphide bonds are preferentially formed in the endoplasmic reticulum
The BACTERIAL PERIPLASM is an oxidizing compartment
(ER)
It can therefore surrogate the ER, although translocating nascent polypeptides through the inner membrane introduces a delicate step
It hosts enzymes catalyzing both disulphide bond formation and isomerization, as well
as specific chaperones and foldases
The number of available gates to the periplasm is limited so that metastable precursors may accumulate in the cytoplasm
The fusion to suitable leader peptides allows to translocate the
unfolded precursors into the periplasm by either
the Sec (post-translational)
or the SRP (co-translational) system
SEC dependent translocation (General Secretory Pathway)
periplasm The Sec(YEG) translocases form a
transmembrane channel
The energy is provided by the ATPase SecA
The co-translational SRPs system utilizes the same channel as the GSP, but instead of SecB, a Small
RibonucleoProtein (SRP) acts as the chaperone
Ffh
5’ 3’
the SecB chaperone binds the pre-protein and brings it to the
general secretion apparatus
SRP binds to the signal peptide of the nascent polypeptide forming an SRP–ribosome nascent chain complex
then binds to its own receptor (FtsY) on the cytoplasmic side of the inner membrane, pulling the complex
FtsY
the SRP pathway is an alternative to the post-translational secretion “SEC” and it is required to avoid premature folding of the proteins in the cytoplasm
Another translocation system is TAT (Twin Arginine Translocase)
the Tat apparatus is energised exclusively by the transmembrane proton electrochemical gradient (Δp)
TAT
It is made of three essential (Tat A, B, C) transmembrane proteins and two accessory
ones (D,E)
The signal peptides which are recognized by TAT are characterized by a couple of arginine residues at the N-terminus
Differently from Sec, TAT translocates only the correctly folded peptides
periplasm
It is about 25 AA long (17-18 25-27)
The typical signal sequence of Gram-negative (didermal) bacteria
positive hydrophobic polar Mature Peptide
THE LENGTH IS CRITICAL
SO AS THE STRUCTURE
At least 1 positively charged residue (Lys Arg) within the first 7 ones
A central hydrophobic region (L V I)
A Ser or Ala rich C-TERMINUS
Frequently Pro at (-6)
Typically an Ala (-3 -1) = cut site for the signal peptidase (Ala-X-Ala) (less frequently Ala is substituted with Ser or Gly
P S S A H A L V L F L A L L Y M K K F
cotranslational translocation by SRP needs the presence of highly hydrophobic leader sequences
N-region H-region C-region
MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATAAQA
This is mainly due to the extended N-region
S/T-R-R-X-F-L-K
To be targeted to the Tat pathway the N-terminal signal peptides must harbour at least two consecutive arginine residues within an S–R–R–x–F–L–K consensus
the H-region of Tat signal peptides is less hydrophobic than that of Sec-specific signal peptides owing to the presence of more glycine and threonine residues
TAT signal sequences are longer than their SEC counterparts (38 aminoacids, on average)
Some of the most popular..
PEL B (PECTATE LYASE)
ST-II (E. coli THERMOSTABLE TOXIN)
DSBA (OXIDOREDUCTASE)
OMPT (MEMBRANE PROTEASE)
K12
ETEC
PEC
PHO A (ALKALINE PHOSPHATASE )
A Sec signal sequence is present in almost all the expression vectors
Recombinant peptide
MRTLTTLGLALLLAQPAVA AQAVLPQLQPYTAPAAWLTPVAPLRIADN
MRTLTTLGLALLLAQPAVAAQA VLPQLQPYTAPAAWLTPVAPLRIADN
? ?
MRTLTTLGLALLLAQPAVAAQAVLPQLQPYTAPAAWLTPVAPLRIADN
http://www.cbs.dtu.dk/services/SignalP/
Any ambiguity in the leader peptide should be avoided
This signal peptide, from the chromosomal b-lactamase B3 of Pseudomonas otitidis showed two possible cutting points
some online services analyze the putative signal sequences and try to predict their efficiency and the most probable cutting site
artificial peptides can be designed
MIA-1/MIA-2: the same peptide but different nucleotide sequence (same CAI value) (aimed to co-express two different peptides on the same vector, avoiding homologous recombination)
MIAmax: the best one..
MIAperC: introduces a cloning NcoI site (CCATGG) without altering the AMA motif for the signal peptidase II
Even when fused with a suitable signal sequence, some large cytoplasmic proteins or some mutants obtained by a combinatorial approach could fail to be
translocated
Secondary/tertiary structure
Chaperones recognizing
attempts to solve the problem could be tried by
Overexpressing the DsbC Disulphide-isomerase
Overexpressing the wide range Skp/OmpH: chaperone
Testing different leader/chaperones combinations
The bias could arise from
To avoid the possible congestion of the translocation systems
It is possible to:
Decrease the expression rate
Increase some limiting components of the translocation system
e.g. introduce plasmid copies of prlA4 e secE genes, encoding the main transport proteins
Once in the periplasm, the folding is enzymatically catalyzed by
With the exception of DsbB, these proteins belong to the thioredoxin protein superfamily
the Dsb oxidases/isomerases
chaperones such as Skp DegP and FkpA
peptidyl-prolyl isomerases such as SurA PpiA and PpiB
. the Dsb protein system (DsbA, B, C, D, G) mediates the disulphide bond
formation and rearrangement
. The formation of disulphide bonds in a protein is made possible by two related pathways:
an oxidative pathway, which is responsible for the formation of the disulphides
OXIDATIVE PATHWAY Disulphides are introduced into the substrate
proteins trough exchanging them with the periplasmic protein DsbA, which is, in turn,
reoxidized by the inner membrane protein DsbB
and an isomerization pathway which shuffles incorrectly formed disulphides
Substrates with more than two cysteines may form incorrect disulphides, causing
them to misfold
THE ISOMERASE PATHWAY
Non-native disulphides are corrected by DsbC and DsbG which are maintained in their active reduced state by the inner
membrane protein DsbD
To facilitate the purification of heterologous proteins it is possible to fuse them with other proteins (fusion partners) or with
short aa stretches (peptide TAGS)
The MCS allows to fuse the TAG in frame with the coding sequence of the recombinant peptide
MCS TAG
5’ FUSION
MCS TAG
3’ FUSION
Once expressed, the recombinant protein has to be purified
Vectors are available that allow to position the tag to the N- or the C- terminal end
Whenever the tertiary structure of the peptide is available, the TAG is placed at the solvent-accessible end
If a signal peptide for the secretion has to be added the TAG is placed at the C-terminal end
TAGS DRAWBACKS
Need for expensive proteases
The cutting efficiency never reaches 100% and limits the yield
frequent need of further treatment (eg. the formation and isomerization of disulphide bridges) in order to obtain an active product
the lack of preliminary indications on the possibility to obtain the solubilization, which can only be determined experimentally
pBR322
pBR322 4361 bp
TET R AM R
pMB1 ori
Rop bom/nic
Designed in 1977 by the scientist Bolivar and Rodriguez, pBR322 has been the first artificial plasmid vector
The mob gene is absent but the nic/bom sites could allow its mobilization by an helper plasmid
POPULAR PLASMID VECTORS FOR E. COLI
ori: pMB1 (belongs to the ColE1 family of plasmids) Selection/screening BLATEM (Tn3) and tet (pSC101)
This plasmid has a low copy number (~20 copies per cell) due to the action of the Rop protein and of RNAI and RNAII
the plasmid replication is initiated by RNAII which hybridizes strongly to the plasmid
The formation of this hybrid at the origin is critical for plasmid replication
RNaseH digests RNA II yielding a 550 nt molecule that acts as a primer for DNA polymeraseI and initiates the replication of the entire plasmid
RNAII
RNaseH
RNAII
primer
replication
RNAI is a non-coding RNA that acts as an antisense repressor of plasmid replication within ColE1 plasmids
RNAI
RNAI concentration increases together with the copy number
RNAI anneals to RNAII by complementary base pairing and blocks the access to RNAseH thus prohibiting RNAII from its initiation role
RNAII
RNAI
This results in a negative feedback loop for replication, setting the average number of plasmids per cell
the plasmid encodes also the Rop (repression of primer) protein
Rop further enhances and stabilizes the interaction
between RNA I and RNA II
RNA-II
RNA-I ROP
RNAI accumulates and hybridizes with RNAII
The plasmid is replicated and the copy number increases
At a low plasmid concentration
RNAII is transcribed from the plasmid
But when the plasmid concentration trespasses a certain treshold
Replication stops and the plasmid copy number
reaches a steady state
The RNAI-Rop regulation prevents the copy number to increase
The deletion of the rop gene, coupled with a point mutation that reduces the
formation of the RNA I/RNA II duplex, led to
The higher copy number of pUC plasmids (derivatives of pMB1 plasmids)
pMB1ori and BLATEM selection are derived from pBR322
pBR322 4361 bp
Puc18/19 Small plasmids with a very high copy number up to 500-700/cell
Plac MCS 5’- -lacZ
The screening marker is lacZ; lacI is not on the vector so that basal activity is high and can be limited in lacIq strains
The absence of both mob and bom/nic ensures that the Puc18/19 can’t be mobilized by helper plasmids
pUC18 and pUC19 differ only for the MCS orientation
Most probably the point mutation hampers the interactions between RNA I and RNA II by producing a temperature-
dependent alteration of the RNA II conformation
pBluescript II SK/KS (+)/(-)
ori: ColE1; selection BLATEM; screening lacZ
Include phage elements PHAGEMIDS
T3 and T7 promoters flank the MCS
Possibility of in vitro transcription by phage polymerases
MCS= KpnI SacI (KS) o SacI KpnI (SK) within the β-galactosidase α-peptide
ColE1
Philamentous phage F1 origin (+) or (-)
The bacterial cells are infected with an M13 mutant phage
(M13K07)
M13K07 copies the ssDNA according to the (+) or (-) orientation
Once packed in the viral particles the ssDNA can isolated to be used as a probe or for site specific mutagenesis techniques
ColE1
lacZ frag
F1- F1+ Makes it possible the phagemid to be
rescued as sense or antisense single-stranded (ss) DNA by an helper phage
(+)
(+)
Infectious form
Replicative form dsDNA
Bidirectional replication
pII inserts a nick in the (+) strand
The rolling circle replication starts
Once completed completed the (+) strand
is cut by pII Is released and circularizes
M13 biological cycle
Host enzymes
Host enzymes
Replicative form
pII mut recognizes only poorly the engineered origin
So the phagemid is preferentially replicated
M13K07 + phagemid
M13K07 is an M13 phage mutated in pII
phagemid
The original replicon of the phage has been modified by several lacZ insertions P-II
mut
And packed into the phage
The PET series
based on PBR322
Expression vectors
Transcription vectors
*** S10 MCS T7-T
T7 SD gene «10» derived from ΦT7
The cloned gene fuses with the N-terminal end of S10; T7 is induced according the features of the host strain
In time other “optional characters" were added to this popular series, changing the selection, adding TAG as histidine strands and / or phage elements
The series includes several vectors
The lowercase letter (a,b,c o d) denotes the reading frame of S10, as referred to the cloning site BamHI, placed
downstream of the signal sequence
a) GGT CGC GGA TCC b) GGT CGG GAT CCG c) GGT CGG ATC CGG d) The same frame as “c” but NcoI (CCATGG) instead of
NdeI (CATATG) upstream to the signal sequence
Selection: AMP other than in the «9»series where it is kan 11: T7/lac + lacI are on the plasmid
12: downstream S10 there is the OmpT signal sequence suitable for the secretion
5: no terminator has been inserted
top related