Increase Your Probability of Success - Lucigen

Increase Your Probability of Successwith Faster, Easier Protein Expression

in E. coli

Overview

• 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

Purification

Common Expression and Purification WorkflowKey challenges at each step

Cloning Expression

• Time-consuming process

• Multiple steps where process can “go wrong”

• Many methods are not amenable to automation

• Poor clone survival

• Low expression levels

• Low solubility (inclusion bodies)

• Protein degradation or truncation

• Poor cell viability with toxic targets

• Non-optimized conditions

• Non-optimized purification strategies

• Occluded purification tags

• “False solubility”• Incomplete fusion

tag cleavage

Characterization

• Truncated protein• Mutated protein• Insufficient yield• Inactive protein

Repeating steps & moving backward is costly

• 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!

Many Parameters to TestExample: trial and error is time-consuming

How Can Protein Expression Go Wrong, and Why?

IssuePossible

ExplanationsPotential Solutions Tools

Low or noexpression

Protein is toxic to E. coli

Rare codons causing translational stalling

Degradation

• Suppress basal induction, tightly control induction level• Reduce plasmid copy number• Express protein in inactive state

• Optimize target sequence• Use strains that supply limited tRNAs• Increase cell density (biomass) before induction• Add easily translatable n-terminal leader sequence

• Slow production, reduce exposure to cellular proteases

• 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

Inclusion body formation

Improper folding causing low solubility

Incorrect disulfide bond formation

• 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

• Direct protein to periplasm• Use host with oxidative cytoplasmic environment

• N-terminal fusion tags• Engineered host strains• Tunable promoters

• Engineered host strains• Periplasmic export fusion

tag

Inactive protein

Mutations in DNA sequence

Incorrect or unremoved tag interferes with activity

Lacking post-translational modifications or cofactors

• Confirm plasmid sequence – promoter + target + fusion• Use recA- strains to maintain stability in cell• Use fresh transformants for expression• Reduce plasmid copy number

• Coexpress transferases, add cofactors• Switch expression host

• recA- host strains• Engineered host strain

(copy control)• N-terminal fusion tags• Protease cleavage• Yeast, insect or

mammalian systems

What strategies do you use,

to express a functional protein

in E. coli?

Choose all that apply.

Question #3Poll Question

How Can the Expresso® Solubility and Expression Screening System Help?

Problem Source Lucigen Solutions

Low SolubilityEnhance 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 ScreeningOptimized 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), primersfor colony screening / sequencing

• Reagents for induction of expressionTarget ProteinTag ENLYFQ G

TEV

Increase Your Probability of SuccessSafeguard 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

– 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

Day 4Day 3Day 2

Solubility & Expression Screening Workflow“Gene to Protein” in as Few as 4 Days

Designone set of PCR primers for each gene of interest.

Amplify your gene(s).

Combine: PCR product(s)+ Expresso vector+ competent cells

Transform E. coli.

Evaluatetarget protein expression and solubility.

Purifyprotein (Ni2+) and remove fusion tags with SelecTEV™ Protease.

Startcultures for expression (same E. coli strain).

Day 1

Characterize solubleprotein.

• 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

pSOL™ Expression VectorsUniversal design allows simplified cloning

Seven Fusion Tags and Control IncludedThree novel tags, four known tags

ONLY kit with a panel of fusion tags

Which solubility / expression -enhancing fusion tags have you

found to be most effective

in E. coli? Choose all that apply.

Question #3Poll Question

Workflow StepsPCR and Cloning (Day 1)1. Amplify gene of interest with 18bp Expresso® overhangs:

2. Check PCR product on agarose gel

3. If PCR reaction yields a single, robust product, combine:

– 2 µL PCR product

– 2 µL pSOL™ Vector

– 1 aliquot (40 µL) 10G Chemically Competent Cells

4. Proceed with transformation and plate on kanamycin plates

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’

Workflow StepsColony 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

24 of 24 colonies correct

Expresso® cloning is highly efficient

1. Start 2 mL overnight cultures from PCR-verified colonies

2. Harvest 1 mL overnight culture, prep DNA and prepare a glycerol stock (optional)

3. Use 30 µL overnight culture to inocculate3 mL of fresh medium

4. Add rhamnose to 0.2% @ OD600 = 0.5

5. Harvest after ~4 hours

• Use lower concentrations of rhamnosefor difficult, insoluble or toxic proteins (0.001% to 0.1%)

Protein Expression Levels are Responsive to Rhamnose Concentrations Between 0.001% & 0.2%

Workflow StepsStandard induction (Days 2 and 3)

Modulate expression levels by varying rhamnose concentrations

Late autoinduction:

Start autoinduction cultures with more glucose (repressor) to delay protein expression

Build up more biomass before induction begins to produce higher yields

Early Autoinduction Late Autoinduction0 6 8 10 24 0 6 8 10 24Hours

• Autoinduction uses glucose to repress the rhaPBAD promoter

• Cells preferentially utilize glucose during early growth

• rhaPBAD promoter is only activated after glucose depletion

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

Alter glucose concentration for toxic targets

AutoinductionMethod

% Glucose

% Rhamnose

Early 0.05% 0.2%

Late 0.15% 0.2%

Workflow StepsAutoinduction (Days 2 and 3)

Rhamnose Promoter Allows Toxic Gene ExpressionLymphostatin (lifA) E. coli virulence factor

• 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

• Generated functional mutants

• Cloned and expressed homolog ToxB in same system4

– Induced with 0.2% rhamnose, 20 hours at 20oC

1. Klapproth, Infect. Immun., 2000

2. Janka, Int.J.Med. Micro. 2000

3. Cassady-Cain, J.Biol.Chem. 2016

4. Cassady-Cain, Infect. Immun, 2017

Figure 1c, LifA, gel and Western blot3

Figure 8, LifA, ToxB4

Workflow StepsEvaluate expression and solubility (Day 3 or 4)

Analysis by SDS-PAGE:• 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

T S I T S I

6xHis

Control

pSol™

Fusion

Fusion Partners Can Rescue Expression & SolubilityExample: 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 ProteinsExample: mammalian nuclease inhibitor

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)

T = total protein, S = soluble protein, I = insoluble protein

Fusion Partners May Help Expression OR SolubilityExample: 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 EfficacyPreferred 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

• 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 literatureto be difficult to express recombinantly.

• Results may differ for different protein targets.

Importance of Screening Multiple Fusion TagsTags Vary in Their Ability to Solve Specific Problems

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Cloning and Transformation OptionsScale for single or multiple targets

10G Chemically Competent Cells, 96-well plates:• 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.

10G Chemically Competent Cells:

• 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.

pSol 1pSol 2pSol 3pSol 4pSol 5pSol 6pSol 7pSol 8

Target 1 2 3 Q-tray

Expresso® Cloning and Expression is ScalableExample: functional annotation

• 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

• Fibrobacter succogines – rumen bacterium that digests cellulose• Whole genome sequencing revealed subset of 48 enzymes of interest

Brumm, Appl Biochem Biotechnol, Aug 2010Suen, PLoS ONE, Apr 2011

• 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]

Successful High-Throughput Screening with Expresso®Test Seven Tags. Re-order the Ones that Work for You.

Custom Solutions

Cloning and Expression WorkflowTips for successCloning:

• 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 WorkflowTips 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 SystemFlexible kit conformations

Cat No. Accura® Polymerase

pSol™ Vectors

E. cloni 10G Cells

SelecTEV™ Protease

49060-1 √ √

49064-1 √ √ √

49062-1 √ √ √

49066-1 √ √ √ √

SelecTEV™ Protease and Accura® Polymerase are available separately.

FailSafe™ PCR SystemReliable 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

Which protease do you prefer, when removing a fusion tag from

your protein of interest? Choose all that apply.

Question #3Poll Question

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 RecoveryMany options for fusion tag removal

ProteaseMolecular

Weight

CleavageRecognition

Site

ResidualN-terminal

Amino AcidsSpecificity

Activity and Reaction

ConditionsNotes

Enterokinase 31 kDa DDDDK / None √√Incompatible with reducing

reagents

Does not cleave proteins Pro in

P1’ position

Thrombin 36.7 kDa LVPR / GS Two: Gly-Ser √No reducing

reagents, 45oC rxn

Verypromiscuous

Factor Xa 43 kDa I(E/D)GR / None √√Incompatible

with EDTA/EGTA

Does not cleave proteins with

Pro or Arg in P1’

SUMO 26 kDatertiary

structureNone √√√

Wide range of pH conditions

Some limitations with residue in

P1’

TEV 27 kDa ENLYFQ / G One: Gly √√√Wide range of

pH and salt conditions

Well-characterized

• 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 ProteaseSimplify tag cleavage with 6xHis-tagged protease

1) Express and purify tagged target protein by IMAC (e.g. Ni2+ column).

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 ProteinProtease

Cleavage Site6xHis

Protease6xHis+

Target Protein

6xHis + Protease6xHis

+ Subtractive IMAC

Target Protein

Protease Cleavage Site

Target Purification and Tag Removal

Purified tagged protein

Untagged protein

Purified untagged protein

6xHis Tagged SelecTEV™ ProteaseEngineered for improved stability and activity

• 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 ProteaseOptimized for stabilized SUMO tag cleavage

• 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

• Exhibits reduced reactivity with native SUMO sequences

• Optimal reaction conditions:

– pH 8.0 (active from pH 6.0 to 10.0)

– 30oC (active from 4oC to 37oC)

– Salt concentrations below 500 mM

– Non-ionic detergents OK at 1%

• Tolerates up to 2 M urea

• Tolerates up to 0.5M guididine HCl

- + P

-, +: with / without SUMO Express Protease

P: purified target protein after subtractive IMAC

SUMO Express and SelecTEV™ ProteasesBest 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 CleavageOptimization

• 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 GuideFusion partner removal for Expresso® systems

Application Requirements

RecommendedSystem

FusionPartners

Terminus Tag Removal?

Protease Included?

Accepts one add’lGly residue on N-terminus

Expresso Solubility and Expression Screening System

6xHis plusFusion partner for expression

& solubility

N-terminus

SelecTEV™ Protease

Optional

6xHis tag acceptable

Expresso T7 Cloning and Expression System

6xHis Choice ofN- or C-

terminus

No No

Native protein ONLY – no extra amino acids

Expresso T7 SUMO Cloning and Expression System

6xHis - SUMO N-terminus

SUMO Express

Protease

Yes

6xHis tag acceptable

Expresso RhamnoseCloning and Expression System

6xHis Choice ofN- or C-

terminus

No No

Native protein ONLY – no extra amino acids

Expresso RhamnoseSUMO Cloning and Expression System

6xHis - SUMO N-terminus

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

Lucigen Tech [email protected](888) 575-9695(608) 831-90118 am – 5 pm Central Time

Thank You!