Download Strategies for Improving Soluble Protein Production in E. coli

Strategies for Improving Soluble
Protein Production in E. coli
Key Learning Objectives
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Overview of recombinant protein expression in E. coli
Challenges in protein expression
Solutions
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Clone quickly
Reduce background expression
Fine-tune expression levels
Overcome solubility challenges
Express difficult membrane proteins
Why Do We Express Recombinant Proteins?
For Research and Commercial Applications
To answer basic biological questions:
• In vivo: Cellular functions and
mechanisms including proteinprotein interactions, temporal
growth studies, signal transduction,
reporter assays
• In vitro: Study protein structure,
function, and activity
To purify proteins for:
• Research products and services
• Industrial enzymes
• Drug discovery
• Biotherapeutics
Applications for Recombinant Proteins
Crystal structure
determination
Enzyme Classification
Industrial enzymes for
consumer products
doi:10.1371/journal.pone.0151001.g004
Drug discovery
Research tools
Biotherapeutics
Human kinases
(>500)
E. coli is Cheapest, Quickest, Easiest System
The Protein Expression Host of Choice based on Pubmed
Proportion of Recombinant Genes Expressed in Different Organisms
Front. Microbiol., 05 March 2014 | http://dx.doi.org/10.3389/fmicb.2014.00085
E. coli Advantages:
• Well-understood genetics, easily manipulated
• Easy introduction of recombinant DNA into cells
• Fast, high density cell growth
• Inexpensive media
• Easy to scale for fermentation
General Protein Expression and Purification Workflow
Cloning Through in vitro Analysis
Cloning
Expression
Purification
Analysis
Key Challenges at Each Step
• Time-consuming
process
• Low expression
levels
• Multiple steps
where process
can “go wrong”
• Low solubility
(inclusion bodies)
• Many methods
are not
amenable to
automation
• Poor clone
survival with
toxic targets
• Protein
degradation or
truncation
• Non-optimized
purification
strategies
• Occluded
purification tags
• Contaminating
proteins
• Poor cell viability
with toxic targets
• Ineffective cell
lysis method
• Need to transfer
vector from
cloning to
expression strain
• Poor salt and
detergent choices
• “False solubility”
• Inactive protein
• Insufficient
protein yield
• Insufficient
protein purity
• Endotoxin
contamination
• Incorrect posttranslational
modifications
Protein Expression in E. coli
Workflow from Gene to Protein
Tag
Promoter
Replication
Origin
Selection
Marker
Clone
Target Gene
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Choose an expression vector
Insert the target gene
Transfer the cloned DNA into the host strain
Express the protein
Evaluate protein yield, solubility and/or activity
Tag
Promoter
Target
Gene
Replication
Origin
Selection
Marker
Transform E. coli
E. coli
Express Protein
Target Gene
Evaluate
Choose an Expression Vector
Vector Determines Downstream Strategy
Tag
Promoter
Selection
Marker
Replication
Origin
 Choice of promoter:
• Inducible or constitutive?
• Requires engineered expression strain?
 Selection marker:
• Ampicillin selection prone to satellite colonies
• Carbenicillin or kanamycin less prone to satellites
 Replication origin:
• High or low-copy
• Inducible copy number
 Fusion tags:
• Purification or detection
• Enhance expression/solubility
• Reporter
Selection
Marker
Replication
Origin
Clone
Target Gene
Tag
Promoter
Target
Gene
Replication
Origin
Selection
Marker
Transform E. coli
E. coli
Express Protein
Target Gene
Promoter
Tag
Evaluate
Insert the Target Gene
Many Cloning Technologies are Available
Tag
Promoter
Replication
Origin
PCR-amplicon
Cloning Workflow
Selection
Marker
Clone
Target Gene
Tag
Promoter
Other Methods:
• Restriction enzyme-based (“cut and paste”)
• LIC (Ligation Independent Cloning)
• In-Fusion®
• TOPO®
• Recombinational (Gateway®)
• Gibson Cloning (SGI-DNA, NEBuilder®)
Target
Gene
Replication
Origin
Selection
Marker
Transform E. coli
E. coli
Express Protein
Target Gene
Estimated preparation time: ~10 hours
Evaluate
Transfer the Clone into E. coli
Different Methods Address Different Needs
Tag
Promoter
Selection
Marker
Replication
Origin
Chemically Competent Cells:
• Easy to work with, less expensive
• No specialized equipment required
• Accept larger DNA volumes
• Lower transformation efficiency than
Electrocompetent cells
• Used for routine cloning and plasmid
propagation
Electrocompetent Cells:
• Shorter protocol, amenable to
automation
• Highest transformation efficiency
• Electroporator instrument required
• Low DNA volumes with no salt required
• Used for library construction and
propagation
Clone
Target Gene
Tag
Promoter
Target
Gene
Replication
Origin
Selection
Marker
Transform E. coli
E. coli
Express Protein
Target Gene
Evaluate
Induce Expression and Produce Protein
Successful Results Depend on this Strategy
Tag
Promoter
Replication
Origin
Key Variables that Determine Success:
 The DNA sequence
• Codon optimized for E. coli
• Optimized for secondary structure
• Target truncation
 Activity of promoter
 Cloning & host strain capabilities
• Toxic genes, repetitive structures
• “Leaky” expression
 Growth & induction conditions
• Cell density at time of induction
• Length and temperature of induction
• Concentration of inducing agent
Selection
Marker
Clone
Target Gene
Tag
Promoter
Target
Gene
Replication
Origin
Selection
Marker
Transform E. coli
E. coli
Express Protein
Target Gene
Evaluate
Protein Expression at the Molecular Level
Many Potential Challenges Exist
Improper
folding
Transmission electron microscopy
Insoluble
Protein
Origins of error:
 Low tRNA availability
 mRNA secondary structure
 Translation rate too slow or
too fast
Toxicity
 Expression level too high
Coupled transcription/translation
Degraded
Protein
What Can Go Wrong and Why?
Issue
Possible Explanations
No or low
expression
Protein may be toxic to cell before
or after induction
Codon bias
Formation of
inclusion
bodies
Inactive
protein
Potential Solutions
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Suppress basal induction of protein- use tight promoters, defined media
Use special engineered hosts
Use tunable promoters
Reduce copy number
Direct protein to periplasm
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Optimize cDNA sequence
Use strains that are capable of supplying limited tRNAs
Increase cell density (biomass)
Use n-terminal fusion tags to overcome translational stalling
Improper folding, low solubility
• Fuse protein to solubility partners
• Co-express molecular chaperones or use folding additives and cofactors to
medium
• Remove inducer and add fresh medium
• Reduce production by modifying RBS, reducing temperature, tuning inducer
concentration
Incorrect disulfide formation
• Direct protein to periplasm
• Use special host with oxidative environment
Missing post-translational
modification
• Use an alternate host
Incomplete folding
• Reduce growth temperature, promote disulfide bond formation
Mutations in cDNA
• Confirm plasmid sequence
• Use recA- strains to maintain stability in cell
• Use fresh transformants
Lacking post-translational
modifications or cofactors
• Coexpress transferases, add cofactors
• Switch to yeast, insect or mammalian cell systems
Solutions to Common Challenges in Cloning
and Expression
Clone with quick, simple HTP-compatible method
 Expresso® Protein Expression Systems
Express high levels of protein with strong E. coli promoters
 Expresso® Rhamnose and T7 Cloning and Expression Systems
Express toxic proteins by tightly controlling expression
 Expresso Rhamnose Cloning and Expression System
Improve protein solubility, express toxic proteins
 Expresso SUMO Systems
 Expresso Solubility and Expression Screening System
Express membrane proteins from T7 promoter
 OverExpress™ C41 (DE3) and C43 (DE3) cell lines
Question #3
Poll Question
What is the native source and
type of the protein(s) you are
trying to express?
Choose all that apply.
General Protein Expression and Purification Workflow
Cloning Through Analysis
Cloning
Expression
Purification
Analysis
Key Challenges at Each Step
• Time-consuming
process
• Low expression
levels
• Multiple steps
where process
can “go wrong”
• Low solubility
(inclusion bodies)
• Many methods
are not
amenable to
automation
• Poor clone
survival with
toxic targets
• Protein
degradation or
truncation
• Non-optimized
purification
strategies
• Occluded
purification tags
• Contaminating
proteins
• Poor cell viability
with toxic targets
• Ineffective cell
lysis method
• Need to transfer
vector from
cloning to
expression strain
• Poor salt and
detergent choices
• “False solubility”
• Inactive protein
• Insufficient
protein yield
• Insufficient
protein purity
• Endotoxin
contamination
• Incorrect posttranslational
modifications
Simple Cloning and Expression Solutions
Speed, Ease & High Efficiency with Expresso® Cloning
• Simple method to clone your PCR product into a Lucigen expression vector
• Uses simple homologous recombination to fuse insert and vector
– Reaction happens inside E. coli cells, during transformation
• Speeds workflows
– Removes PCR product clean-up and ligation steps
– Reduces pipetting steps: compatible with automated screening platforms
• Directional cloning
• No cloning scars
• Uses pre-processed,
linearized vector
• Highly efficient
Colony PCR Shows >90% Correct Clones
Expresso® Cloning in Three Simple Steps
Instant Cloning by in vivo Homologous Recombination
1. Amplify target by PCR
• Primers include ~18bp
overlap with Expresso vector
sequence
2. Mix PCR product and
Expresso vector with
competent cells
3. Transform and plate cells
normally
Expresso® Workflow Comparison
Fastest and Easiest PCR-based Cloning System Available
• No vector preparation
• No restriction enzymes or
ligase needed
• No DNA purification steps
• Fewer handling and
pipetting steps
• Compatible with
automation
Expresso® Cloning Primer Design
Design PCR Primers with Overlapping Sequences
Forward primer:
5’ – CAT CAT CAC CAC CAT CAC – 18 – 24 nucleotides gene specific sequence
Reverse primer:
5’ – GTG GCG GCC GCT CTA TTA – 18 – 24 nucleotides reverse compliment gene specific sequence
Expresso® Systems are Complete Kits
Expresso Cloning and Protein Expression Systems include:
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Linearized, dephosphorylated expression vector
Competent cells for cloning and expression
Control insert for cloning, expression and protease cleavage
Primers for sequencing
Sugar solutions for induction
Protease enzymes for fusion tag cleavage
Poll Question
Which challenges are you facing in
your protein expression projects?
Choose all that apply.
General Protein Expression and Purification Workflow
Cloning Through Analysis
Cloning
Expression
Purification
Analysis
Key Challenges at Each Step
• Time-consuming
process
• Low expression
levels
• Multiple steps
where process
can “go wrong”
• Low solubility
(inclusion bodies)
• Many methods
are not
amenable to
automation
• Poor clone
survival with
toxic targets
• Protein
degradation or
truncation
• Non-optimized
purification
strategies
• Occluded
purification tags
• Contaminating
proteins
• Poor cell viability
with toxic targets
• Ineffective cell
lysis method
• Need to transfer
vector from
cloning to
expression strain
• Poor salt and
detergent choices
• “False solubility”
• Inactive protein
• Insufficient
protein yield
• Insufficient
protein purity
• Endotoxin
contamination
• Incorrect posttranslational
modifications
Common Protein Expression Challenges and Solutions
Key Roadblocks to Soluble, Active protein
Degradation
Toxicity
Codon Bias
Inclusion
Body
Formation
http://biosocialmethods.isr.umich.edu/epigenetics-tutorial/epigenetics-tutorial-gene-expression-from-dna-to-protein/
How Can the Expresso® T7 Cloning and Expression
System Help?
Challenge
Lucigen Solutions
Toxicity
Control basal expression with HI-Control™ cells.
Low Solubility
Enhance solubility with optional SUMO tag.
Low Expression
Produce high levels of protein from inducible T7
promoter.
Time-Consuming
Cloning
Save time and increase throughput with Expresso
cloning.
High-level, Inducible Expression with Low Background
Optimized for Tight Induction Control
Achieve High Expression Levels
• IPTG-inducible T7 promoter
Choose Optimized Vectors
• N-or C-terminal 6xHis tag
• Small: ~2.2kb
• Kanamycin resistance
• Transcriptional terminators
• Optional SUMO tag
T
Transcriptional terminators stabilize clones by preventing
toxicity due to transcription into and out of cloned fragments
Reduce Background Expression
• HI-Control™ cells reduce “leaky” expression during cloning and expression
• Facilitates production of toxic proteins
Reduce Problematic “Leaky” Expression
Clone and Express Challenging Targets with HI-Control™
Problem:
Background expression of T7 RNA
Polymerase
= Uninduced target gene expression
Decreased colony count and cell viability
during cloning and expression
Solution:
HI-Control BL21(DE3)
HI-Control Cells Express LacIq Repressor
Lac
Repressor
LacUV5 Promoter
Lac Operator
T7 RNA Polymerase
T7 Promoter
Lac Operator
Target Gene
+ IPTG
HI-Control 10G (cloning strain)
Induced Target
Gene Expression
Increased expression of LacIq repressor
= Improved control of gene expression
Less toxicity and more soluble protein
T7 RNA
Polymerase
Lac Operator
Target Gene
Ideal System for Routine or Toxic Proteins
High-level Expression Equivalent to the pET System
 Induce maximal protein
expression from strong T7
promoter
 Control “leaky” expression with
strains expressing high levels of
lac repressor (LacIq):
 HI-Control™ 10G
 HI-Control BL21 (DE3)
 Purify protein with 6xHis tag
 Clone simply with Expresso®
cloning
How Can the Expresso® Rhamnose Cloning and
Expression System Help?
Challenge
Toxicity
Lucigen Solutions
Control basal expression and fine-tune induction level of
toxic proteins with tunable rhamnose-inducible
promoter.
Enhance solubility with optional SUMO tag.
Low Solubility
Low Expression
Time-Consuming
Cloning and Transfer
Find optimal induction conditions with tunable
rhamnose promoter to maximize soluble protein yield.
Find optimal protein expression conditions using tunable
rhamnose promoter.
Save time and increase throughput with Expresso
cloning.
Save time by cloning and expressing in the same strain.
Tunable Protein Expression with Virtually No Background
Ideal System for Toxic Proteins
Fine-Tune Expression Levels
• Rhamnose-inducible rhaPBAD promoter
• Enhanced solubility
Select Optimized Vectors
• N-or C-terminal 6xHis tag
• Small: ~2.3kb
• Kanamycin resistance
• Transcriptional terminators
• Optional SUMO tag
Transcriptional terminators stabilize clones by preventing
toxicity due to transcription into and out of cloned fragments
Express Toxic Proteins
• Eliminate “leaky” expression with glucose repression
• Express proteins in almost any E. coli cell line
Control the Level of Protein Expression
Maximize Soluble Protein Yields
 Save time by using a single strain for
cloning AND expression
 Eliminate “leaky” expression with glucose
repression
 Find the best conditions for induction of
toxic proteins
 Easily modulate expression levels by varying
rhamnose concentrations (standard
induction protocol)
 Combine glucose and rhamnose in the
growth media for (hands-free
autoinduction)
 After glucose is depleted from media,
cells switch to rhamnose as a carbon
source
 Rhamnose-induced protein expression
begins
Protein Expression Levels are Responsive to
Rhamnose Concentrations Between 0.001% &
0.2%
Control the Timing of Protein Expression
Alter Glucose Conc. for Toxic Target Autoinduction
Hours
Early Autoinduction
0
6
8
10 24
Late Autoinduction
0
6
8 10 24
Late autoinduction:
 Start autoinduction cultures with more glucose (repressor) to delay protein expression
 Build up more biomass before induction begins
 Produce higher protein yields
Question #3
Poll Question
Which fusion tags have you used
to express your protein in E. coli?
Choose all that apply.
Improve Protein Expression and Solubility
Express Soluble, Native Proteins with Expresso® SUMO
Trusted SUMO Fusion Technology
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Small Ubiquitin-like Modifier (100 amino
acid yeast protein)
Enhance functional protein production
with N-terminal SUMO tag
Reduce likelihood of inclusion bodies (i.e.
insoluble protein)
Recover Native Protein
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Cleave SUMO tag using SUMO Express
Protease
Easily remove protease with Ni2+ column
purification
Select Your Promoter
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Expresso T7 SUMO Cloning and Expression System – for high-level production
Expresso Rhamnose SUMO System – for controlled, tunable production
Recover Native Proteins Quickly
Simplify Purification and Tag Cleavage
1) Express and purify tagged
target protein by IMAC
(Ni2+ column).
6xHis
+
2) Add SUMO Express
Protease (with 6xHis tag)
and incubate.
3) Remove 6xHis-SUMO tag
and SUMO Express
Protease by subtractive
IMAC (Ni2+ column).
4) Purified, native protein is
recovered from the
column flow-through.
SUMO
Tag
6xHis
SUMO
Tag
+
Target Protein
SUMO
Protease
6xHis
Target Protein
+
Target Protein
+
6xHis
Subtractive
IMAC
SUMO
Protease
Increase Soluble Protein Yield
Cleavable SUMO Tag Enhances Expression and Solubility
Gene 1
Gene 2
SUMO tag enhances solubility
compared to c-terminal 6xHis
tag alone
Suen (2011) PLoS ONE 6(4):e18814.
Low Recombinant Protein Solubility?
Evaluation and Next Steps
M T
S
I
Analysis by SDS-PAGE:
• Grow E. coli clone(s) 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.
Potential Solutions for Insoluble Proteins
Impact
Purify protein as inclusion bodies. Denature and
re-fold protein.
Methods are problematic. Requires high degree of skill.
Optimize protein induction parameters, export.
Time-consuming, may not work.
Use optimized system with fusion partners to
enhance solubility and expression levels.
Screen multiple fusion tags simultaneously with tunable
promoter, easy expression protocol and fast cloning method.
How Can the Expresso® Solubility and Expression
Screening System Help?
Problem Source
Lucigen Solutions
Low Solubility
Enhance solubility with a panel of fusion tags.
Low expression due to protein
or mRNA degradation
Stabilize mRNA and protein with fusion tags.
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.
Toxicity
Tightly control expression levels with
rhamnose promoter.
Use a high-throughput compatible cloning
Need for multiplex capabilities and screening workflow. Save time by using a
single strain for cloning and expression.
Enhance Expression and Solubility with Fusion Partners
Seven Expresso® Vectors Contain a Panel of Fusion Tags
Test your amplicon with all tags in parallel:
ONLY kit with a panel
of fusion tags
 Improve expression with tunable promoter
 Produce native protein with SelecTEV™-cleavable tags
 Purify native protein from 6xHis-SelecTEV™ Protease
Expression & Solubility Screening Workflow
“Gene to Protein” in as Few as 4 Days
Day 1
Design
one set of
PCR
primers to
amplify
your gene.
Day 2
Amplify your gene.
Clone your PCR product
into all seven Expresso®
fusion vectors plus the
control vector.
Transform E. coli.
Start cultures
for
expression
(same strain).
Day 3
Evaluate
target
protein
expression
and
solubility.
Day 4
Purify
protein (Ni2+)
and remove
fusion tags
with
SelecTEV™
Protease.
Soluble,
native
protein.
High Efficiency Speeds Workflows, Enables Automation
Clone Into 7 Expresso® Vectors with >90% Efficiency
24 / 24 Correct Clones by Colony PCR Screen of SOL-LIN28 Target
Streamline time and effort
with high efficiency cloning:
 Eliminate repeat cloning
attempts
 Pick fewer colonies for
analysis
 Perform fewer minipreps,
sequencing
 Reduce pipetting steps,
high-throughput
compatible
Quickly Identify the Best Solubility Tag for Your Protein
Streamline Screening with Parallel Processing
slyD
AFV
T
S
I
T
*
S
tsf
I
T
*
S
I
T
*
S
MBP
Bla
SUMO
I
T
S
*
I
T
*
S
Control
GST
I
T
*
S
I
T
S
*
T = total protein, S = soluble protein, I = insoluble protein
SMAD protein: Recruited to the TGF-b receptors and mediates TGF-b signaling
Results: Six of seven tags showed enhanced solubility compared to control (Lane S).
Four of seven tags showed enhanced expression compared to control (Lane T), with
Tsf and MBP as best performers.
I
*
Different Tags Rescue Different Proteins
Best Tag for Each Protein is Determined Empirically
T = total protein, S = soluble protein, I = insoluble protein
GH1 is the form of human growth hormone expressed in the pituitary gland.
Results: SlyD, Tsf, SUMO, Bla, and MBP demonstrated enhanced solubility (Lane S),
compared to control.
SlyD, Tsf, Bla and MBP also demonstrated enhanced expression (Lane T).
Successful High-Throughput Screening with Expresso®
Test Seven Tags. Re-order the Ones that Work for You.
• Clone the 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
Improve Expression of Toxic/Membrane Proteins
OverExpress® C41(DE3) & C43(DE3) Competent Cells
 Mutant strains derived from
BL21 (DE3) for toxic proteins
(membrane proteins,
cytoplasmic proteins,
nucleases)
Comparison of OverExpress with BL21(DE3)
 Proven success with over
350 referenced publications
 Reduced expression from T7
promoters
 Strains differ in tolerance for
different proteins
 Also available as pLysS for
tighter expression control
Dumon-Seignovert, et al. , (2004). Protein Expression and Purification 37, 203-206.
Literature references: http://www.lucigen.com/OverExpress-References.html
Basic Tips and Troubleshooting
•
Design cloning primers carefully
– Check reading frame, start and stop codons, and cloning sequences
•
Sequence-verify all expression clones
– Lucigen kits include sequencing primers!
– Troubleshooting: sequence-verify target in plasmid purified from expression strain
•
•
Test more than just one colony for expression
Sonicate cells to evaluate total protein expression (instead of lysing cell pellet in SDSPAGE loading buffer)
– Without sonication, overexpressed protein may complex with DNA = viscous, hard-to-load
sample
– The target protein may not enter the gel and won’t be visualized on the gel
•
•
Try tunable promoter for difficult targets (Expresso® Rhamnose System)
Optimize expression temperature and timing
– Express at 30oC or 22oC to slow down protein production
– Try different autoinduction protocols (late induction vs early induction)
•
•
Try alternative tags, add tags to opposite termini
Move to alternative E. coli strain
Summary
If You Need To……
Express
high levels
of protein
Expresso® T7 Cloning and
Expression System
√
Expresso® T7 SUMO Cloning and
Expression System
√
Express an
insoluble
protein
Express
membrane
proteins from
T7 promoter
√
Expresso Rhamnose SUMO
Cloning and Expression System
OverExpress™ C41(DE3) and
C43(DE3) competent cells
Improve or
optimize
expression
conditions
√
Expresso Rhamnose Cloning and
Expression System
Expresso Solubility and
Expression Screening System
Express a
toxic
protein
√
√
√
√
√
√
√
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