Download Heterologous expression and purification of proteins in E. coli

Heterologous expression and
purification of proteins in E. coli
Rory Koenen
Institut für molekulare Herz-Kreislaufforschung
University Hospital of the RWTH Aachen
[email protected]
Tel. 35984
contents
Things to consider when planning to express proteins in E. coli
An introduction to protein purification
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http://www.imcar.rwth-aachen.de/
Click „Verschiedenes“ and then „Lehrmaterial“
Protein Expression
prokaryotic gene expression
• expression vector features
• bacterial host features
• solubility
• affinity tags
• general considerations
Features of expression vector
Origin: Essential for plasmid propagation.
High copy vs low copy.
Examples are pBR322, ColE1, pACYC
T7 promotor/operator:
Transcription initiation site for T7 RNA polymerase.
Operator is binding site for Lac repressor in the absence of lactose.
Ribosome binding site:
needed for initiation of translation
Multiple cloning site:
facilitates cloning of the desired cDNA
Resistance marker:
essential for plasmid propagation
Lac Repressor:
needed for control of transcription and expression of cDNA
not essential if the bacterial host already has a LacI gene
Features of expression vector
Origin
T7 promotor/operator
Ribosome binding site
MCS
Lac Repressor
Expression vector
2961 bp
Resistance Marker
Novagen pET26b
Features of bacterial hosts
T7 RNA polymerase gene:
Viral RNA polymerase that has high rates of transcription and
is not needed for endogenous transcription of bacterial genes.
Bacterial hosts that have the (DE3) lysogen are competent
for T7-based vectors
Some sophisticated strains have the T7 gene under LacI control
Protease-deficient:
some proteases degenerate the expressed protease.
The BL21 strain is deficient for OmpT and Lon proteases.
E. coli B or K strain-derived:
can sometimes make a difference...
Gene mutations for „oxidative cytoplasm“:
may influence disulfide bond formation in some proteins
Presence of the Lac Repressor gene LacI:
needed for control of transcription and expression of cDNA
Expression
• cells are grown typically until mid-log phase
• induction occurs by the lactose analog isopropyl-thiogalactoside:
IPTG: non-metabolized lactose analog
• Cells are harvested and prepared for purification
Expression
• protein can occur as soluble native protein (uncommon)
• or as insoluble aggregates (common)
• these insoluble aggregates are generally „stored“ in E. coli as
so called inclusion bodies
• inclusion bodies are dense and can be purified easily by
centrifugation
• they are very immunogenic and can be readily injected in
animals
• BUT they need to be dissolved, and the protein refolded to the
native state
• refolding can be very problematic
solubilization and refolding
• inclusion bodies can be dissolved using:
– detergents (e.g. laurylsarcosine)
– chaotropic salts (ureum, guanidine-HCl, arginine)
– organic solvents or high/low pH buffers
• refolding can take place by:
– strong dilution in „native“ buffer
– dialysis in „native“ buffer
– binding to a column and perfusion with „native“ buffer
Disulfides can be formed by additives like:
–
–
–
–
–
cysteine – cystine pair
reduced and oxidized glutathione
copper and o-phenanthroline
hydrogen peroxide
just air
affinity/solubility tags
•
greatly simplify purification; especially for beginners/ non-experts
•
the alltime classic is the 6*histidine tag, which can be purified using
metal chelation chromatography
•
another classic is the glutathione-S-transferase tag which binds
strongly to GST-sepharose
•
other tags are chitin binding protein, maltose binding protein, polyarginine and many more.
•
solubility tags increase solubility of protein in cytoplasm:
– examples are: thioredoxin, Nus.
protein
His
His
His
His
His
His
+
Affinity chromatography
resin
resin
affinity tags
•
the His-tag is a good choice because:
-
is often functionally neutral: no need for removal
-
metal chelation chromatography is cheap, easy and permits denaturing conditions
-
may support on-column refolding
BUT
-
choice of column buffers is limited
-
protein mostly not so pure after single affinity chromatography step
•
the GST-tag is also good because:
-
it facilitates the fusion of small peptides
-
sometimes supports solubility of protein
-
a single purification step generally leads to very pure protein
BUT
-
slow kinetics of binding
-
columns are expensive
-
cleavage often required but even more often quite problematic
-
GST very immunogenic and stable
general considerations
•
N-terminus or C-terminal tags
– what is known about protein function?
– modified N-terminus can have big consequences: Met-RANTES
– use a cleavable tag with a good protease like enterokinase or TEV
•
Codon usage
– eukaryotic genes have different codon occurance as prokaryotic genes: Pro
CCC and Arg AGA are common in humans but rare in E. coli
•
Protein toxicity / plasmid stability
– toxic proteins do not express well and the E.coli cell will try to shut the
expression down, sometimes by destroying the plasmid
– even worse, during the growth, cells that express even traces of toxic
protein will die, leaving you with cells that do not express anything
– „leaky expression“ can be decreased using pLysS or pLys E plasmids in
the host
Protein Purification
protein purification
• Chromatography:
– affinity: Ni-NTA, protein G/A (antibodies), GST-sepharose
– kation exchange: positive charge by e.g.
SP sepharose, Mono S
– anion exchange: negative charge by e.g.
Q sepharose, Mono Q
– gel filtration: separation by size, Superose, Superdex, Sephacryl
– hydrophobic interaction and reverse phase HPLC: hydrophobicity
protein purification
• ion exchange chromatography
– separation by charge
– protein charge depends on buffer pH
– elution from column by pH shifts or shifts in
ion strength (preferred)
– permits a lot of different conditions
– choice number one for routine purifications!
Ion-exchange chromatography
protein purification
• gel filtration chromatography
– separation by size
– biggest proteins come first, smallest come last
– permits a lot of different buffer conditions
BUT
– GF columns are expensive and fragile
– sample size should be very small (<1% of
column volume)
– not a very good „first step“, more suitable for
final refinement
Gel filtration chromatography
protein purification
• reverse phase chromatography
– separation by hydrophobicity
– elution using organic solvents
– high resolution and very quick
– removes endotoxin contaminations
– eluted proteins can immediately be analyzed
using mass spectroscopy
BUT
– some proteins do not survive the buffer
conditions
– best performed on a HPLC system, which is
an investment
Purification of human PF4 from E.coli
Slides: Alisina Sarabi
expression of recombinant PF4
• plating of E. coli containing vector
– Rosetta2(DE3) and pET26b/PF4
• growth in specialized medium
• induction by IPTG and expression
overnight
• Centrifugation of the cells
• Storage of pellet at -30°C
Purity
Purification strategy
3.Polishing
High level purity
2.Intermediate
purification
Remove bulk
impurities
1.Capture
Isolate, concentrate,
stabilize
Preparation,
Extraction,
clarification
Step
SP Sepharose
Capture
Buffer A: 50mM NaAc pH 5,5
Buffer B: A + 2M NaCl pH 5,5
SP Sepharose Flow through
After dialysis against 50mM NaAc pH5,5
the crude periplasmic solution containing
the protein was purified by FPLC (0-2M
NaCl gradient) using a HiLoad 16/10 SPSepharose™
Intermediate
MonoS
Buffer A: 50mM NaAc pH 5,5
Buffer B: A + 2M NaCl pH 5,5
CaptoS
Buffer A: 50mM NaAc pH 5
Buffer B: A + 2M NaCl pH 5
Pool A13-B13
After dialysis against 50mM NaAc (pH 5,5 or 5) the protein was purified by FPLC (0-2M
NaCl gradient) using a using a strong cation exchanger (MonoS™ 5/50 GL or HiTrap CaptoS)
Polishing
Reverse phase chromatograpy
After dialysis 2 x against
1% acetic acid and 1 x
against 0,1% TFA protein
was purified by using a
Recource™ RPC column
Buffer A: 0,1% TFA
Buffer B: 0,1% TFA + 90% ACN
Coomassie staining
Pool B8+B7
Polishing
HPLC: Hypersil Gold (C-18)
Silverstaining SDS Page
Western Blot
Mass spec (esi)
PF4alt
experiment. mass: 7805.8 D
theoret. mass:
7806.1 D
Thank you for your attention