Expression & Purification of
Recombinant Proteins
August 22, 2011
Biochemistry 201
D. Worthylake, 7152MEB, x5176
Why express/purify protein(s)?
1) If you don’t have the gene that encodes the protein
but you have a source, you may want to purify the
protein to:
a) determine the amino acid sequence
b) make antibodies
c) Identification by mass spectroscopy
2) If you have the gene that encodes the protein, you may
want to express/purify the protein for other reasons:
a)
b)
c)
d)
e)
f)
structural analysis (x-ray crystallography & NMR spectroscopy)
enzyme function
Interaction partners
biochemistry/biophysics (phosphorylation, regulation, etc.)
Functional studies (cellular localization by confocal microscopy, etc)
Pharmaceutical intervention
TOP 10 Things to consider
1)
Which protein construct to express
2)
Expression host (bacterial, insect cell, yeast, or mammalian)
3)
Cell line for expression
4)
Promoter for induction of protein production
5)
Codon optimization (for mammalian proteins expressed in bacteria)
6)
Cloning method
7)
May require expression as a fusion protein
8)
May require co-expression with molecular chaperones
9)
Affinity tag(s) for purification & protease cleavage site to remove the tag
10) Purification protocol & buffer to keep protein happy and active
Protein construct and expression host
1) Engineering of protein construct
a) An entire protein ?
If yes, don’t need to worry about limits
b) A domain from a mosaic protein ?
Need to worry about limits
2) Which organism to express the protein in
a) If protein is of bacterial origin, express in bacteria
b) If protein is of non-bacterial origin, because of post-translational
modification in non-bacterial cells, may need to express in higher
organisms:
Bacteria  Yeast  insect cells (SF9 or Hi5)  Mammalian cell lines
(least expensive &
(most expensive &
time consuming)
time consuming)
c) May need to express as a fusion protein or require codon-optimization
Choice of expression host
Bacterial expression system
Advantages –
Easy, great over-expression, low protease activity,
no post-translational modifications
Disadvantages – Protein solubility, lack of post-translational modifications
Eukaryotic expression system
Advantages –
Protein solubility, post-translational modifications
Disadvantages – Expensive, low yield, proteases, time consuming
Isolate protein from native source
Advantages –
Protein solubility, authenticity
Disadvantages – Expense/effort, yield, slaughter-houses
Waring blenders
Hierarchy: Bacteria, Yeast, SF9, Hela, native tissue
Bacterial host BL21(DE3)
Hosts for protein expression
a) DE3
1)
2)
3)
4)
Host-encoded lac Repressor represses host
RNAPol transcription of T7 RNAPol from lac
promoter.
IPTG induction knocks lac Repressor off and
allow host RNAPol to transcribe T7RNAPol.
T7 RNAPol transcribe gene from T7 promoter
on plasmid.
Lon/OmpT protease deficient.
b) DE3_pLysS
1)
2)
DE3 strains have leaky expression, which
leads to problems if expressed protein is toxic.
Plasmid-encoded T7 lysozyme inhibits T7
RNAPol and decrease leaky expression.
c) Host for cloning
1)
These hosts lack T7 RNAPol , and so are
suitable for plasmid amplification and not
protein expression.
Isopropyl β-D-1-thiogalactopyranoside (IPTG) = allolactose mimic
Figures from www.novagen.com
Specialized bacterial cell lines for protein expression
Company
Invitrogen
Clontech
Cell
BL21Star
BL21-AI
Rosetta2
Origami 2/B
BL21Pro
Genotype
RNaseE(131)
ara7 rare tRNA
Thrx-/GluthRedTetR
Phenotype
more stable mRNA
arabinose induction
overcome codon usage deficiency
enhance formation of disulfide bonds
tetracycline induction
Lucigen
C41/C43
??
Expression of toxic proteins
T7 lysozyme
decreases leaky expression
Novagen
all companies pLysS
p15A
p15A
p15A
F/p15A+F
p15A
p15A
p15A
You cannot just mix and match, as plasmids with the same origin of replication cannot be
transformed into the same cells. Most expression plasmids have pBR322 ori, which is
compatible with p15A.
Consideration of codon usage
Basic elements of a plasmid/vector
pET developed by WF Studier & BA Moffatt
in 1986
1)
Ap = ampicillin resistance
2)
ori = ColE1/pBR322 origin of replication
3)
lacI = lac repressor; bind lacO until IPTG
induction
4)
T7P = T7 Polymerase promoter
5)
lacO = lac operator where lac
repressor binds
6)
= multiple cloning site
Selection of cloning method is critical
1.
Restriction digestion-based methods are inefficient and require that your gene of
interest does not have the same internal restriction site(s) as present in MCS.
2.
Gateway-based methods are powerful.
3.
Ligation-independent cloning is much more effective than ligation reaction.
The green/red parts of the
primers are not
complementary with the gene!
Gateway technology overview (discussed for completeness)
See file invitrogen_Gatewaymanual2003.pdf (optional reading)
Gateway recombination reactions
Entry
clone
Expression
clone
See file invitrogen_Gatewaymanual2003.pdf (optional reading)
Generating an entry clone
TAGG
ATCC
(Original site)
*
(Original site)
Don’t put in stop codon
(*) if to have a C-ter tag
(Int + IHF)
TAGG
ATCC
*
See file invitrogen_Gatewaymanual2003.pdf (optional reading)
Recombining entry and destination clones to get an
expression clone
TAGG
ATCC
*
(Int + IHF + Xis)
TAGG
ATCC
*
(Original site restored)
(Original site restored)
See file invitrogen_Gatewaymanual2003.pdf (optional reading)
PCR and Processive DNAPol
new
Cycle 1: 2o = 1
cumulative
1 = 21 - 1
Cycle 2: 21 = 2
2 + 1 = 3 = 22 - 1
www.neb.com
Cycle 3: 22 = 4
4 + 3 = 7 = 23 - 1
Cycle n: 2n-1
2n - 1
…
15 cycles =
32,767 copies
20 cycles =
1,048,575 copies
30 cycles = 1,073,741,823 copies
1)
2)
3)
decreases lower-molecular weight fragments
decreases extension time from 1’/kb to 10-20”/kb, so a
typical amplification takes ~1hr instead of 3-4 hours.
NEB Phusion (dbd) & Takara Speedstar (antibody-based)
LIC cloning, reminder
3.
Ligation-independent cloning is much more effective than ligation reaction.
LIC-Subcloning of a gene, from beginning to end
DAY 1 (~6.5 hr)
1. PCR gene from SOURCE vector (50l) (80’)
2.
Verify on agarose gel that gene was amplified (10l) (40’)
3.
Digest remaining sample with DpnI and do PCR clean-up (75’)
4.
Digest DESTINATION vector and PCR fragment with T4 DNAPol
to generate single-stranded overhangs (60’)
5.
Add 100ng PCR fragment to 15ng DESTINATION vector and
let anneal on ice (30’)
6. Transform into XL10Gold supercompetent cells (100’)
and plate overnight
DAY 2 (~2.5 hr)
7. Do colony PCR on 2-4 colonies to verify gene was inserted
into vector:
a) pick colony with pipet tip
b) resuspend in 50l steril water and vortex to mix
c) take 1l for PCR to verify insert (120’)
d) inoculate remaining sample in TB for overnight growth
e) if gene is inserted, do miniprep (30’)
f) Verify construct by DNA sequencing
Bacterial transformation
1.
Frederick Griffith (1928)
first demonstrated by showing that non-virulent Streptococcus pneumonia could be made
virulent by exposing it to a virulent strain which has been heat-shocked.
2.
Oswald Avery (1944)
demonstrated uptake of DNA into bacteria and coined the term bacterial transformation.
3.
To make cell competent:
Grow cells to mid-log phase and treat/wash with CaCl2 solution. The bacterial cell wall is
permeabilized by Chloride ion and swells up with the uptake of water.
4.
a)
Bacterial transformation:
Addition of plasmid DNA to cells, followed by cold- and heat-shock allows plasmid to enter
through the small holes in the cell wall. (Can also use electroporation to create pores in cell
wall).
b) Amplify the number of cells in SOC media.
c) Plate on LB agar (+ antibiotic) to select for transformed cells only (antibiotic resistance is
conferred by gene encoded by on plasmid).
Selection of expression vector & fusion partner
 Expression in insect and mammalian cells are expensive and time consuming.
 Therefore, a feasible method is required for expression in bacterial cells as a
first choice.
 Expression difficulty in bacterial cells may be overcome by expression as a
fusion protein. Expression at lower temperature improves solubility.
Laila Niiranen, … Nils P. Willassen, Protein Expression & Purification 52 (2007) 210-218.
 The apparent solubilizing effect of the fusion partner may be misleading,
as the purified protein can precipitate when cleaved from its fusion partner.
LIC vector for in-vivo cleavage from fusion protein
In-vivo cleavage helps remove false-positive expression (protein once
purified and cleaved from its fusion partner precipitates!).
Co-express TVMV protease with fusion protein, with TVMV under control
of a different promoter than the one used for the fusion protein.
In-vivo cleavage improves protein solubility
Soluble
insoluble
In-vivo cleavage helps protein purification
Intact fusion protein
in-vivo cleaved protein
M L FT W E +tev
L FT W E +tev
L FT W
L FT W
Delayed In-vivo cleavage improves protein solubility
0hr delay
2hr delay
Co-expression with molecular chaperones
Trigger factor (Tf)
• binds 50S subunit;
• peptidyl-prolyl cis-trans isomerase
DnaJ/K
• binds nascent polypeptides;
• shield exposed hydrophobic patches
from folding unfavorably
GrpE
• binds polypeptides released from
DnaK/J
• releases polypeptides into folded form
or shuttles to GroEL/ES
GroEL/ES
• helps fold/refold proteins already in
compact state but are not yet folded
Learn all you can before beginning
MSA can often give you ideas for deciding on construct limits
Even better if there’s some structural information!
If multiple sequence alignments do not help and there
isn’t any structural info, try secondary structure prediction
..but try several starts
and stops (primers are
cheap!)
http://www.compbio.dundee.ac.uk/www-jpred//
Do you have the gene?
Lots of output!
Here’s what you want
buy
Shopping cart – price varies with order size
Before starting, confirm that you can make a significant quantity of
soluble protein. Small scale solubility experiments are very
important and typically will involve varying inducer concentration,
expression temperature, expression construct, etc.
Each protein is unique – must exploit differences
Particular affinities
GST, 6xHis, antibodies
Solubility
(NH4)2SO4, PEG precip.
Charge
ion exchange
Hydrophobicity
hydrophobic chromatography
Size
gel exclusion
Iso-electric point
iso-electric focusing
Thermal stability
alter temp.
Nickel-affinity chromatography (Histrap)
Express protein in frame with an affinity tag – often tag is removable with a protease.
Common tags: 6xHis, GST, CaM, MBP. Use affinity chromatography for first step!
electron coordination bonds
Imidazole
Nitrilotriacetic acid
pH  7.4
Kirkegaard & Perry Laboratories, Inc
If the affinity tag is removable, go back over column and collect
flow-through (or digest on the column).
Ion exchange chromatography (what is the theoretical pI of your protein?)
DiEthylAminoEthane (DEAE), CarboxyMethyl (CM), Quaternary amine, Sulfonic
acid.
http://www.proteinchemist.com/tutorial/iec.html
These functional groups are charged over a broad pH range. Why would that be
desirable?
Anion exchange chromatography
Anion #2
( Cl- )
-
Na+
+
pH=6
+
+
+
+
+
+
+
Bind (Low salt)
-
+
Na+
YFP
YFP
Anion #1
( protein )
Cl-
+
+
Na+
Cl-
+
+
+
Na+
Cl-
+
Na+
Cl-
Elute (High salt)
+
Run a 20 x (column volume) linear gradient and collect fractions
500mM NaCl
Linear gradient
(also step)
Trp, Tyr, Phe,
disulfides
50mM NaCl
example chromatogram
Run SDS-PAGE of fractions to decide which to pool
(sacrifice yield for purity?)
Stronger and higher resolution ion exchange media (Q, SP) may
be employed to separate proteins that were not baseline
separated with weak ion exchange step.
Some proteins, usually larger proteins, can bind to
both anion and cation exchange matrices – change
pH to enhance interaction.
Electrostatic potential mapped
onto a molecular surface
Q column
SP column
Size exclusion chromatography
Separates proteins by size.
Your protein should elute at the proper volume for its expected MW.
Want a nice, symmetric peak in the chromatogram.
Small proteins “see” a
bigger volume than do
large proteins
Some other chromatographic techniques
Salting out – Proteins precipitate differentially in the presence
of (NH4)2SO4 or polyethylene glycol - It’s probably worth trying
Hydrophobic – Load proteins onto phenyl sepharose in
presence of ~1.5M (NH4)2SO4 and run decreasing [(NH4)2SO4]
gradient. More hydrophobic elutes later.
Isoelectric focusing – Electrophorese protein in matrix containing
pH gradient. When the protein reaches that pH where it has no net
charge it ceases to migrate. Retrieve protein from matrix.
Expression of TVMV protease
Day 1
Transform Rosetta 2 cells with plasmid containing tvmv gene and plate overnight
Day 2 (or -1)
In the evening, pick a colony and grown a 10ml overnight starter culture @ 37C/200RPM.
(you can save a day by inoculating from a glycerol stock.)
Day 3 (or 1)
a) In morning, inoculate 2L media with overnight starter and grow @37C/200PRM.
b) After 2-4 hours (OD600 ~0.6-1), add IPTG to 0.5mM final concentration and induce for
4hrs @30C/200RPM.
Expression optimization:
at mid-log phase, lower temperature to 12-20C, add lower amount of IPTG and induce
overnight, or for slow leaky expression, no IPTG for 2 days (membrane proteins)
c) Harvest cells (4000 RPM/20’), resuspend in 50mL lysis buffer (+ protease inhibitors) and
store in -80C.
Day 4 (or 2)
a) thaw cells from -80C and lyse by sonication or with Emulsiflex (cells are squeeze through
a small pin-hole by high pressure).
b) Start purification
Purification of TVMV protease
1)
2)
3)
Load sample in 10mM Imi (pH8)
Wash 5CV of 10mM Imi in 1M NaCl (helps remove DNA
bound to DNA-binding proteins)
Start gradient:
a) 1030mM / 5CV (initial wash)
b) 3060mM / 30CV (more stringent wash)
c) 60500mM / 1CV (start elution)
d) 500mM / 5CV (complete elution)
Purification of TVMV protease
1) Nickel column
Pool for S75
2) Superdex 75 26/60
Vo (~110ml)
Injection
save
Confirming expressed protein by Western
Anti-His6 western
Western blotting
1) 30-45’/210V: run SDS-PAGE gel
[SDS binds protein tightly (1 SDS/2 aa) to give
equivalent q/m ratio for all proteins; hence proteins are
separated based on MW]
1) 2x 15’: rinse gel in TB
2) 45’/35V: electroblot (shown to the left)
3) 30’: wash NC in 25ml PBST + 5% w/v fat-free milk
4) 3x 5’: wash NC in 25ml PBST
5) 30’: soak NC in 20ml PBST + antibody_HRP (1:20000)
6) 3x 5’: wash NC in 25ml PBST each
7) 5’: expose NC to substrate, 0.75 + 0.75 ml
(Pierce SuperSignal West Dura)
9) Develop/image NC
~3.5 hours total
TB: 25mM Tris[8.3], 192mM Glycine)
PBS: 1.54mM KH2PO4, 2.71mM Na2HPO4, 167mM NaCl
PBST: PBS + 0.05% v/v Tween20)
10 things you should know
1) How BL21(DE3) cells work (host-encoded T7RNAPol and lacI repressor)
2) General idea about different bacterial cell lines for optimizing expression:
a) different promoters
b) increasing mRNA stability
c) Lon/OmpT protease deficiency
d) Rosetta2 cells and codon-optimization
3) Basic idea of plasmid and antibiotic selection.
4) Different cloning method (ligation, LIC, Gateway)
5) How PCR works – use of processive DNAPol to save time
6) Bacterial transformation
7) Improving protein solubility by expression as fusion protein, and which partner works best.
8) in-vivo cleavage of protein from fusion partner
9) co-expression with molecular chaperones
10) Basic idea of protein purification
a) nickel-affinity (Histrap)
b) Size-exclusion
c) ion-exchange
d) western blot
e) hydrophobic