Increase Your Probability of Success
with Faster, Easier Protein Expression
in E. coli
Overview
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•
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•
•
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Common challenges when expressing recombinant proteins in E. coli
Solutions and tools to help mitigate those challenges
Examples & data
Scaling your workflow for one or many targets
Proteases for fusion tag removal
Tips & best practices
Common Expression and Purification Workflow
Key challenges at each step
• Time-consuming
process
• Multiple steps
where process can
“go wrong”
• Many methods are
not amenable to
automation
• Poor clone survival
Cloning
• Low expression
levels
• Low solubility
(inclusion bodies)
• Protein degradation
or truncation
• Poor cell viability
with toxic targets
• Non-optimized
conditions
Expression
• Non-optimized
purification
strategies
• Occluded
purification tags
• “False solubility”
• Incomplete fusion
tag cleavage
Purification
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•
•
•
Truncated protein
Mutated protein
Insufficient yield
Inactive protein
Characterization
Repeating steps & moving backward is costly
Many Parameters to Test
Example: trial and error is time-consuming
• Two novel sequences from a rare microorganism
• Some homology to a known enzymes family, but you have few clues as to
how well they will express in recombinant form
• You decide to express in E. coli and hedge your bets by trying a few
different vectors and strains available in your lab
– 2 inducible promoters of different strengths
– 4 fusion partners that have worked with other targets
– 2 expression strains
• Two sequences x 2 promoters x 4 tags x 2 strains = 32 samples
• Expands:
– +/- induction
– Total, soluble, insoluble
– other parameters
• Experiments can get big, very fast!
How Can Protein Expression Go Wrong, and Why?
Issue
Low or no
expression
Inclusion
body
formation
Inactive
protein
Possible
Explanations
Potential Solutions
Protein is toxic to E. coli
• Suppress basal induction, tightly control induction level
• Reduce plasmid copy number
• Express protein in inactive state
Rare codons causing
translational stalling
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•
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•
Degradation
• Slow production, reduce exposure to cellular proteases
Improper folding causing low
solubility
• Fuse protein to solubility partners
• Co-express molecular chaperones or add folding
additives and cofactors to medium
• Reduce production: modify RBS, tightly control induction
level, reduce temperature, tune inducer concentration,
inhibit T7 RNA polymerase
Incorrect disulfide bond
formation
• Direct protein to periplasm
• Use host with oxidative cytoplasmic environment
Mutations in DNA sequence
Incorrect or unremoved tag
interferes with activity
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Lacking post-translational
modifications or cofactors
• Coexpress transferases, add cofactors
• Switch expression host
Optimize target sequence
Use strains that supply limited tRNAs
Increase cell density (biomass) before induction
Add easily translatable n-terminal leader sequence
Confirm plasmid sequence – promoter + target + fusion
Use recA- strains to maintain stability in cell
Use fresh transformants for expression
Reduce plasmid copy number
Tools
• Tunable promoters
• Engineered host strain
(copy control)
• Defined media, with
glucose as carbon source
• N-terminal fusion tags
• Engineered host strains
• Protease inhibitors
• Engineered host strains
• N-terminal fusion tags
• Engineered host strains
• Tunable promoters
• Engineered host strains
• Periplasmic export fusion
tag
• recA- host strains
• Engineered host strain
(copy control)
• N-terminal fusion tags
• Protease cleavage
• Yeast, insect or
mammalian systems
Question #3
Poll Question
What strategies do you use,
to express a functional protein
in E. coli?
Choose all that apply.
How Can the Expresso® Solubility and
Expression Screening System Help?
Problem Source
Lucigen Solutions
Low Solubility
Enhance solubility with a panel of N-terminal
fusion tags shown to increase solubility.
Low expression due to codon
bias
Help overcome codon bias at the 5’ end of
sequence with N-terminal fusion tags. Codon
bias at 5’ end often stalls translation.
Low expression due to protein
or mRNA degradation
Stabilize mRNA and protein with fusion tags.
Low or no expression due to
toxicity
Tightly control expression levels with
rhamnose promoter.
Inactive or incorrect protein
due to mutations, deletions,
rearrangements
RecA- strain used for cloning and expression.
Expresso® Solubility and Expression Screening
Optimized system for cloning and expression
• Accura® High-Fidelity Polymerase for
amplifying your target
• Seven ready-to-use pSOL™ Vectors,
each with a different fusion tag proven
to enhance expression and solubility
• E. cloni 10G Chemically Competent
Cells for cloning and expression (same
recA- strain)
• SelecTEV™ Protease for fusion tag
removal
• Control insert (human GH1), primers
for colony screening / sequencing
• Reagents for induction of expression
TEV
Tag
ENLYFQ G
Target Protein
Increase Your Probability of Success
Safeguard your project from the very beginning
• Increase likelihood of initial success
– Screen 7 fusion partners to increase chances of soluble protein production
– Use recA- strain to decrease recombination events
• Scale the process for multiple targets
– Minimize manipulation steps with easy-to-use protocol
– Adapt cloning and expression easily to HTP methods
– Utilize hands-free autoinduction during screening
• Save time
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–
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Design one set of universal PCR primers
Transform only once, use the same strain for cloning and expression
Clone into ready-to-use vectors, without PCR product clean-up
Clone without enzymes or incubations
• Optimize as you like
– Fine-tune expression with highly responsive promoter
– Adjust autoinduction timing and levels
Solubility & Expression Screening Workflow
“Gene to Protein” in as Few as 4 Days
Day 1
Design
one set of
PCR
primers
for each
gene of
interest.
Amplify your gene(s).
Combine: PCR product(s)
+ Expresso vector
+ competent cells
Transform E. coli.
Day 2
Start
cultures for
expression
(same E. coli
strain).
Day 3
Evaluate
target
protein
expression
and
solubility.
Day 4
Purify
protein (Ni2+)
and remove
fusion tags
with
SelecTEV™
Protease.
Characterize
soluble
protein.
pSOL™ Expression Vectors
Universal design allows simplified cloning
• Screen seven fusion partners
with one amplicon
• Small size ~2.3kb (base
vector)
• RhaPBAD promoter allows
tunable expression
• 6xHis tag for purification
• Protease cleavage site for tag
removal
• Kanamycin selection reduces
satellite colonies
• Low copy for increased
stability
Seven Fusion Tags and Control Included
Three novel tags, four known tags
ONLY kit with a panel
of fusion tags
Question #3
Poll Question
Which solubility / expression enhancing fusion tags have you
found to be most effective
in E. coli?
Choose all that apply.
Workflow Steps
PCR and Cloning (Day 1)
1.
Amplify gene of interest with 18bp Expresso® overhangs:
5’-AAT CTG TAC TTC CAG GGT XXX XXX XXX XXX XXX XXX …-3’
5’-GTG GCG GCC GCT CTA TTA XXX XXX XXX XXX XXX XXX …-3’
2.
3.
Check PCR product on agarose gel
If PCR reaction yields a single, robust product, combine:
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–
–
4.
2 µL PCR product
2 µL pSOL™ Vector
1 aliquot (40 µL) 10G Chemically Competent Cells
Proceed with transformation and plate on kanamycin plates
Workflow Steps
Colony screen and overnight cultures (Day 2)
1. PCR screen colonies with universal primers provided
– For long targets, use 1 vector primer & 1 target specific primer
2. Start overnight cultures from positives
17 of 18 colonies correct
Expresso® cloning is
highly efficient
24 of 24 colonies correct
Workflow Steps
Standard induction (Days 2 and 3)
Modulate expression levels by
varying rhamnose concentrations
1. Start 2 mL overnight cultures from PCRverified colonies
2. Harvest 1 mL overnight culture, prep DNA
and prepare a glycerol stock (optional)
3. Use 30 µL overnight culture to inocculate
3 mL of fresh medium
4. Add rhamnose to 0.2% @ OD600 = 0.5
5. Harvest after ~4 hours
•
Use lower concentrations of rhamnose
for difficult, insoluble or toxic proteins
(0.001% to 0.1%)
Protein Expression Levels are Responsive to
Rhamnose Concentrations Between 0.001% & 0.2%
Workflow Steps
Autoinduction (Days 2 and 3)
Alter glucose concentration
for toxic targets
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Autoinduction uses glucose to
repress the rhaPBAD promoter
Cells preferentially utilize glucose
during early growth
rhaPBAD promoter is only activated
after glucose depletion
Early Autoinduction Late Autoinduction
Hours 0 6
8 10 24 0 6 8 10 24
1. Start 4-5 mL overnight cultures
from colonies with glucose and
rhamnose
2. Harvest 1 mL overnight culture for
DNA prep and glycerol stock
(optional)
3. Harvest 10 OD600 units of overnight
culture for expression analysis
Autoinduction
Method
%
Glucose
%
Rhamnose
Early
0.05%
0.2%
Late
0.15%
0.2%
Late autoinduction:
 Start autoinduction cultures with more glucose (repressor) to delay protein expression
 Build up more biomass before induction begins to produce higher yields
Rhamnose Promoter Allows Toxic Gene Expression
Lymphostatin (lifA) E. coli virulence factor
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Produced by most enteropathogenic and
enterohemorrhagic E. coli
Required for host intestinal colonization
Inhibits proliferation of T cells
365 kD (one of the largest bacterial toxins known)
Unstable in recombinant form, low expression1,2
Successfully produced with rhamnose promoter3
– Glucose repression at 37oC to OD600 = 0.8
– Induced with 0.2% rhamnose, 3 hour induction, 30oC
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Generated functional mutants
Cloned and expressed homolog ToxB in same system4
– Induced with 0.2% rhamnose, 20 hours at 20oC
1.
2.
3.
4.
Klapproth, Infect. Immun., 2000
Janka, Int.J.Med. Micro. 2000
Cassady-Cain, J.Biol.Chem. 2016
Cassady-Cain, Infect. Immun, 2017
Figure 1c, LifA, gel
and Western blot3
Figure 8,
LifA, ToxB4
Workflow Steps
Evaluate expression and solubility (Day 3 or 4)
pSol™
Fusion
T
S
I
6xHis
Control
T
S
Analysis by SDS-PAGE:
•
I
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Grow E. coli clones containing recombinant gene of
interest, induce expression
• Take sample of uninduced cell culture, if
applicable
Harvest cells by centrifugation
Resuspend cell pellet in sonication buffer
• Choice of protease inhibitors
• Include reducing reagent (DTT)
• Keep purification requirements in mind
Lyse cells by sonication
• Take sample of Total protein
Fractionate lysate by centrifugation
• Take sample of Soluble protein from supernatant
• Resuspend Insoluble pellet in SDS-PAGE sample
buffer
Analyze fractions by SDS-PAGE and determine which
fraction(s) contain your protein of interest.
Compare solubility and expression to the control (6xHis
ONLY) sample
Determine which pSol Fusion clone to proceed to tag
removal and protein characterization experiments
Fusion Partners Can Rescue Expression & Solubility
Example: GH1 (human growth hormone)
T = total protein, S = soluble protein, I = insoluble protein
Results:
• SlyD, Tsf, SUMO, Bla, and MBP enhanced solubility (Lane S), compared to control
• SlyD, Tsf, Bla and MBP also enhanced expression (Lane T)
Different Tags Rescue Different Proteins
Example: mammalian nuclease inhibitor
T = total protein, S = soluble protein, I = insoluble protein
Results:
• Tsf and MBP provided modest solubility improvement (Lane S), compared to control
• AFV, slyD, Tsf, SUMO, Bla, MBP and GST improved expression (Lane T)
• Co-migrating E. coli bands obscure AFV and slyD solubility results (Lane S)
Fusion Partners May Help Expression OR Solubility
Example: AflIII (restriction enzyme)
T = total protein, S = soluble protein, I = insoluble protein
Results:
• AFV, slyD, Tsf, SUMO and MBP enhanced expression (Lane T), compared to control
• No tags significantly enhanced solubility (Lane T)
Panel of Tags Helps Identify Common Problems
Example: DNA modification enzyme
T = total protein, S = soluble protein, I = insoluble protein
Results:
• Tsf and MBP enhanced solubility (Lane S), compared to control
• No tags significantly enhanced solubility (Lane T)
• Rapid degradation to smaller product seen with all tags and control
Evaluating Fusion Partners for Efficacy
Preferred tags were selected from a larger panel
• In one experiment, 8 GOI’s were tested with 12 fusion partners
• Each combination was evaluated for solubility and expression enhancements
• Tags that performed well moved forward in development
Importance of Screening Multiple Fusion Tags
Tags Vary in Their Ability to Solve Specific Problems
In Order of Best Results
• Proteins react differently to different fusion tags
• In our experience with 24+ targets tested internally, some Expresso® fusion
tags were better at solving expression challenges:
• These results are based on a subset of proteins known in the literature
to be difficult to express recombinantly.
• Results may differ for different protein targets.
Cloning and Transformation Options
Scale for single or multiple targets
10G Chemically Competent Cells,
96-well plates:
10G Chemically Competent Cells:
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Thaw cells on ice (plate can be divided into 4
x 24 wells)
Add:
– 2 µl (25 ng) Expresso® pSOL vector
– 1 µl (at least 5 ng) PCR product
Cover and incubate 20 minutes on ice
Heat shock 20 sec at 42°C (thermal cycler)
Incubate on ice, 1 minute.
Add 180 µl of room-temperature Recovery
Medium and mix.
Cover plate and incubate at 37°C for 1 hour
(thermal cycler). Shaking is not necessary.
Plate kanamycin selective media.
pSol 1
pSol 2
pSol 3
pSol 4
pSol 5
pSol 6
pSol 7
pSol 8
Target 1 2 3
Q-tray
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Thaw cells on ice
Transfer cells to a 15 mL polypropylene tube
Add:
– 2 µl (25 ng) Expresso® pSol vector
– 1-3 µl (25 - 100 ng) PCR product
Incubate 30 minutes on ice
Heat shock 45 sec at 42°C (water bath)
Incubate on ice, 2 minutes.
Add 960 µl of room-temperature Recovery
Medium and mix.
Cover plate and incubate at 37°C for 1 hour
with shaking, 250 rpm.
Plate on kanamycin selective media.
Expresso® Cloning and Expression is Scalable
Example: functional annotation
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Fibrobacter succogines – rumen bacterium that digests cellulose
Whole genome sequencing revealed subset of 48 enzymes of interest
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96-well PCR amplification and cloning
96-well expression induction
96-well assay directly on induced lysates
Also generated shotgun expression library
in Expresso® vector, screened for enzyme
activity
Over 400 proteins produced
Brumm, Appl Biochem Biotechnol, Aug 2010
Suen, PLoS ONE, Apr 2011
Successful High-Throughput Screening with Expresso®
Test Seven Tags. Re-order the Ones that Work for You.
• Clone your target(s) into seven fusion tag vectors, plus the control vector,
which contains a 6xHis tag only.
• Compare protein expression and solubility between the control vector
and the seven fusion tags.
• If you find that some tags work better than others for your proteins, we
will provide those specific Expresso® fusion tag vectors as custom
products (at larger scales, if desired).
• Contact [email protected]
Custom
Solutions
Cloning and Expression Workflow
Tips for success
Cloning:
• Avoid background in your cloning step. If the gene to be amplified is
carried on a kanamycin-resistant plasmid:
– Gel purify the PCR product before cloning
– Treat the PCR product with Dpn1 before cloning
– Linearize the template plasmid with a restriction enzyme
• Use chemically competent cells. Expresso cloning is not compatible with
electrocompetent cells.
• Maximize your transformation efficiency. Transfer the cells + DNA to 15 mL
polypropylene culture tubes before heat shock for optimal heat transfer.
Expression Screening:
• Measure the growth rate of your transformed strain (before and after
induction) vs the growth rate of the parental, untransformed strain.
– If the growth rate changes significantly after induction, you may be seeing
toxicity.
Expression and Expression Workflow
Tips for success
Expression Screening:
• If you suspect toxicity:
– Try a gradient of rhamnose concentrations
– Perform an autoinduction with increased amounts of glucose to increase cell
density prior to induction
– Lower growth temperature (20oC to 30oC) – note that slowing cell growth rate
may require longer induction protocols
• If the protein is insoluble:
– Decrease rhamnose concentration, 0.001% to 0.1%
– Lower the growth temperature
• Assay your protein as early in the process as you can.
• Unlike T7 promoters, the rhamnose promoter is active and inducible in
almost any E. coli strain.
– Clones can be transferred to other favorite strains for expression studies (BL21
or other ompT strain)
Summary
Expresso® Solubility & Expression Screening System
Feature
Problem Solved
Tight control of background expression, reducing
toxicity
Increases expression levels
Tunable expression controls toxicity resulting from
overexpression
Increases expression levels and
protein solubility
Novel and known solubility tags
Enhances protein solubility
N-terminal fusion tags help to overcome codon bias
and enhance protein folding
May increase expression levels
Panel of vectors allows screening of multiple tags
and custom formatting
Choice of effective fusion tag(s) as a
custom product
Single E. coli strain for cloning and expression
(Lucigen E. cloni® 10G Chemically Competent Cells)
Simple workflow, saves time
Optional autoinduction protocol
Simple workflow
Fusion tags are cleavable with SelecTEV™ Protease
Facilitates downstream analysis and
characterization
Expresso® Solubility & Expression Screening System
Flexible kit conformations
Cat No.
Accura®
Polymerase
49060-1
49064-1
49062-1
49066-1
√
√
pSol™
Vectors
E. cloni 10G
Cells
SelecTEV™
Protease
√
√
√
√
√
√
√
√
√
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SelecTEV™ Protease and Accura® Polymerase are available separately.
FailSafe™ PCR System
Reliable hi-fi amplification for every template
• Twelve 2x high fidelity PCR PreMixes
• Each PreMix contains dNTPs, buffer, different concentrations of Mg+2 and
Epicentre's proprietary PCR Enhancer (with Betaine)
• Perform PCR reactions to find the PreMix that amplifies your target
Amplification of an 80%-85% GC-rich
region of the human fragile X gene.
Protease Tag Cleavage
Question #3
Poll Question
Which protease do you prefer,
when removing a fusion tag from
your protein of interest?
Choose all that apply.
When Should Fusion Partners Be Removed?
It’s all about your downstream application
• Many proteins retain structure & function with a fusion partner, but results
are hard to predict
• Crystallography: large fusion partner removal is recommended, but peptide
tag removal may not be necessary
• Enzymology, biochemical or other activity assays: remove fusions whenever
possible
– Assay the protein first – if it’s active in your application, tag removal
may not be necessary
• Large-scale production, therapeutic use: tag removal is often recommended
• Know your termini:
Intended Use of Fusion
Terminus
Solubility or expression enhancements
N-terminus
Fusion must be removed
N-terminus
Affinity purification or detection
N- or C-terminus
Fusion Partner Cleavage and Protein Recovery
Many options for fusion tag removal
Protease
Enterokinase
Thrombin
Factor Xa
SUMO
TEV
Molecular
Weight
31 kDa
36.7 kDa
Cleavage
Recognition
Site
DDDDK /
LVPR / GS
43 kDa
I(E/D)GR /
26 kDa
tertiary
structure
27 kDa
ENLYFQ / G
Residual
N-terminal
Amino Acids
None
Two: Gly-Ser
None
None
One: Gly
Specificity
Activity and
Reaction
Conditions
Notes
√√
Incompatible
with reducing
reagents
Does not cleave
proteins Pro in
P1’ position
√
No reducing
reagents, 45oC
rxn
Very
promiscuous
√√
Incompatible
with EDTA/EGTA
Does not cleave
proteins with
Pro or Arg in P1’
√√√
Wide range of
pH conditions
Some limitations
with residue in
P1’
√√√
Wide range of
pH and salt
conditions
Wellcharacterized
• P1’ position: amino acid immediately following the cleavage site
• Larger aromatic amino acids (e.g. Proline) at this position can cause steric hindrance
• Amino acids with smaller side chains are preferred (Gly, Ser)
SelecTEV™ and SUMO Express Protease
Simplify tag cleavage with 6xHis-tagged protease
Target Purification and Tag Removal
1) Express and purify tagged
target protein by IMAC (e.g.
Ni2+ column).
Purified
tagged
protein
6xHis
Protease
Cleavage Site
+
2) Dialyze or desalt to remove
imidazole.
3) Add 6xHis-tagged Protease
and incubate.
4) Remove 6xHis-SUMO tag and
SUMO Express Protease by
subtractive IMAC.
5) Recover purified protein from
the column flow-through.
Target Protein
Untagged
protein
6xHis
Protease
6xHis
Protease
Target Protein
Protease
Cleavage Site
+
+
Purified
untagged
protein
6xHis
Subtractive
IMAC
Target Protein
6xHis Tagged SelecTEV™ Protease
Engineered for improved stability and activity
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•
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Tobacco Etch Virus (TEV) protease
Recognizes Glu-Asn-Leu-Tyr-Phe-Gln / Gly
Active from pH 6.0 to 8.5
Optimum temperature 30oC, active at 4oC to 30oC
Tolerates up to 0.5M Urea
Cleavage and Purification of GH1
1, 2: Before and after
SelecTEV cleavage
3, 4: Subtractive IMAC.
Column flow through and
wash, showing untagged GH1
5: Column elution, showing
fusion tag and 6xHis-tagged
protease.
6xHis Tagged SUMO Express Protease
Optimized for stabilized SUMO tag cleavage
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SUMO Express is specifically engineered to cleave the modified SUMO tag in the
Expresso® SUMO Cloning and Expression kits
Modified SUMO tag is resistant to eukaryotic desumoylation enzymes
Stabilized tag allows expression of SUMO fusion proteins in insect or mammalian
Transfer your fusion to a mammalian system and retain the benefits of the SUMO
tag
- + P
Exhibits reduced reactivity with native SUMO sequences
Optimal reaction conditions:
– pH 8.0 (active from pH 6.0 to 10.0)
-, +: with / without
– 30oC (active from 4oC to 37oC)
SUMO Express
Protease
– Salt concentrations below 500 mM
P: purified target
– Non-ionic detergents OK at 1%
protein after
Tolerates up to 2 M urea
subtractive IMAC
Tolerates up to 0.5M guididine HCl
SUMO Express and SelecTEV™ Proteases
Best practices
•
When possible, choose a protease with a molecular weight significantly different
from your protein, so you can easily check for tag removal by SDS PAGE.
•
TEV protease tolerates protease inhibitors PMSF and AEBSF.
•
Use proteases at the recommended concentration first, then optimize if needed.
Using higher concentrations can lead to nonspecific cleavage.
•
Optimize the temperature and length of incubation. Remove samples at various
time-points and analyze them by SDS- PAGE.
•
When calculating column capacity for subtractive IMAC, remember to account for
the 6xHis-tagged fragments released by protease incubation, which will also bind
to the resin.
•
DTT can cause leaching from some nickel resins. Test your IMAC resin to ensure
compatibility with the DTT concentration in the recommended protease reaction
buffer. If needed, dilute your sample before loading to reduce DTT concentration.
Fusion Tag Cleavage
Optimization
•
Incomplete tag cleavage:
– Add more enzyme
– Incubate for longer time period (e.g., 4oC overnight)
– Dialyze your protein to remove potential inhibitors (interfering detergents,
imidazole)
•
‘False solubility’ = removing the tag leads to precipitated protein.
– More common with larger (>100 KD) proteins, or proteins with large
hydrophobic patches
– Test other fusion constructs that exhibit apparent solubility
– Vary pH and salt conditions during purification and after tag cleavage
– Use a buffer that differs from the pI by +0.5 pH units (avoid pH <6.0)
Expression System and Protease Guide
Fusion partner removal for Expresso® systems
Application
Requirements
Recommended
System
Fusion
Partners
Terminus
Tag
Removal?
Protease
Included?
Accepts one add’l
Gly residue
on N-terminus
Expresso Solubility and
Expression Screening
System
6xHis plus
Fusion partner
for expression
& solubility
Nterminus
SelecTEV™
Protease
Optional
6xHis tag
acceptable
Expresso T7 Cloning
and Expression System
6xHis
Choice of
N- or Cterminus
No
No
Native protein
ONLY – no extra
amino acids
Expresso T7 SUMO
Cloning and Expression
System
6xHis - SUMO
Nterminus
SUMO
Express
Protease
Yes
6xHis tag
acceptable
Expresso Rhamnose
Cloning and Expression
System
6xHis
Choice of
N- or Cterminus
No
No
Native protein
ONLY – no extra
amino acids
Expresso Rhamnose
SUMO Cloning and
Expression System
6xHis - SUMO
Nterminus
SUMO
Express
Protease
Yes
Resources
Poster: http://bit.ly/expresso-poster
Nature Methods:
http://bit.ly/nature-methods-expresso-rhamnose
http://bit.ly/nature-methods-expresso
Application Notes:
http://bit.ly/expresso-app-note
Questions? www.lucigen.com
Thank You!
Lucigen Tech Support
[email protected]
(888) 575-9695
(608) 831-9011
8 am – 5 pm Central Time