Strategies for Improving Soluble Protein Production in E. Coli Key Learning Objectives

Strategies for Improving Soluble Protein Production in E. coli Key Learning Objectives

• Overview of recombinant protein expression in E. coli • Challenges in protein expression • Solutions • 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 protein- protein 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 Enzyme Classification Crystal structure determination 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 • Low expression • Non-optimized • Inactive protein process levels purification strategies • Insufficient • Multiple steps • Low solubility protein yield where process (inclusion bodies) • Occluded can “go wrong” purification tags • Insufficient • Protein protein purity • Many methods degradation or • Contaminating are not truncation proteins • Endotoxin amenable to contamination automation • Poor cell viability • Ineffective cell with toxic targets lysis method • Incorrect post- • Poor clone translational survival with • Need to transfer • Poor salt and modifications toxic targets vector from detergent choices cloning to expression strain • “False solubility” Tag Protein Expression in E. coli Promoter

Workflow from Gene to Protein Selection Marker Replication Origin

Clone Target Gene

Tag • Choose an expression vector Promoter Target Gene • Insert the target gene Selection Marker Replication • Transfer the cloned DNA into the host strain Origin • Express the protein • Evaluate protein yield, solubility and/or activity Transform E. coli

E. coli

Express Protein

Target Gene

Evaluate Tag Choose an Expression Vector Promoter

Vector Determines Downstream Strategy Selection Marker Replication Origin  Choice of promoter: • Inducible or constitutive? Clone Target Gene • Requires engineered expression strain?  Selection marker: Tag Promoter Target • Ampicillin selection prone to satellite colonies Gene Selection • Carbenicillin or kanamycin less prone to satellites Marker Replication  Replication origin: Origin • High or low-copy • Inducible copy number Transform E. coli  Fusion tags: • Purification or detection E. coli • Enhance expression/solubility • Reporter Express Protein

Target Gene Selection Replication Marker Origin Promoter Tag

Evaluate Tag Insert the Target Gene Promoter Many Cloning Technologies are Available Selection Marker Replication PCR-amplicon Origin Cloning Workflow Clone Target Gene

Tag Promoter Target Gene Other Methods: Selection • Restriction enzyme-based (“cut and paste”) Marker Replication • LIC (Ligation Independent Cloning) Origin • In-Fusion® • TOPO® Transform E. coli • Recombinational (Gateway®) E. coli • Gibson Cloning (SGI-DNA, NEBuilder®)

Express Protein

Target Gene

Estimated preparation time: ~10 hours Evaluate Tag Transfer the Clone into E. coli Promoter

Different Methods Address Different Needs Selection Marker Replication Origin Chemically Competent Cells: • Easy to work with, less expensive Clone • No specialized equipment required Target Gene • Accept larger DNA volumes Tag • Lower transformation efficiency than Promoter Target Electrocompetent cells Gene Selection • Used for routine cloning and plasmid Marker Replication propagation Origin

Electrocompetent Cells: Transform E. coli • Shorter protocol, amenable to

automation E. coli • Highest transformation efficiency

• Electroporator instrument required Express Protein • Low DNA volumes with no salt required • Used for library construction and propagation Target Gene

Evaluate Tag Induce Expression and Produce Protein Promoter Successful Results Depend on this Strategy Selection Marker Replication Origin

Key Variables that Determine Success: Clone Target Gene  The DNA sequence • Codon optimized for E. coli Tag Promoter Target • Optimized for secondary structure Gene

Selection • Target truncation Marker Replication  Activity of promoter Origin  Cloning & host strain capabilities • Toxic genes, repetitive structures Transform E. coli • “Leaky” expression E. coli  Growth & induction conditions

• Cell density at time of induction Express Protein • Length and temperature of induction • Concentration of inducing agent Target Gene

Evaluate Protein Expression at the Molecular Level Many Potential Challenges Exist

Improper Insoluble folding Protein

Transmission electron microscopy Origins of error:  Low tRNA availability  mRNA secondary structure Toxicity  Translation rate too slow or too fast  Expression level too high

Coupled transcription/translation Degraded Protein What Can Go Wrong and Why? Issue Possible Explanations Potential Solutions

No or low Protein may be toxic to cell before • Suppress basal induction of protein- use tight promoters, defined media expression or after induction • Use special engineered hosts • Use tunable promoters • Reduce copy number • Direct protein to periplasm

• Optimize cDNA sequence Codon bias • Use strains that are capable of supplying limited tRNAs • Increase cell density (biomass) • Use n-terminal fusion tags to overcome translational stalling Formation of Improper folding, low solubility • Fuse protein to solubility partners inclusion • Co-express molecular chaperones or use folding additives and cofactors to bodies 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 Inactive Incomplete folding • Reduce growth temperature, promote disulfide bond formation protein • Confirm plasmid sequence Mutations in cDNA • Use recA- strains to maintain stability in cell • Use fresh transformants

Lacking post-translational • Coexpress transferases, add cofactors modifications or 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 Poll QuestionQuestion #3

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

• 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 Colony PCR Shows >90% Correct Clones • Uses pre-processed, linearized vector • Highly efficient 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: • 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

Degradation Toxicity

Inclusion Codon Bias 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.

Produce high levels of protein from inducible T7 Low Expression promoter.

Time-Consuming Save time and increase throughput with Expresso Cloning 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 Transcriptional terminators stabilize clones by preventing T • Optional SUMO tag 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™

HI-Control Cells Express LacIq Repressor Problem: Background expression of T7 RNA Polymerase Lac = Uninduced target gene expression Repressor

Decreased colony count and cell viability LacUV5 Promoter Lac Operator T7 RNA Polymerase during cloning and expression

Solution: T7 Promoter Lac Operator Target Gene HI-Control BL21(DE3) HI-Control 10G (cloning strain) + IPTG Increased expression of LacIq repressor Induced Target = Improved control of gene expression Gene Expression T7 RNA Polymerase Less toxicity and more soluble protein 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 Lucigen Solutions Control basal expression and fine-tune induction level of Toxicity toxic proteins with tunable rhamnose-inducible promoter. Enhance solubility with optional SUMO tag. Low Solubility Find optimal induction conditions with tunable rhamnose promoter to maximize soluble protein yield.

Find optimal protein expression conditions using tunable Low Expression rhamnose promoter.

Save time and increase throughput with Expresso Time-Consuming cloning. Cloning and Transfer 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 Express Toxic Proteins toxicity due to transcription into and out of cloned fragments • 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 Protein Expression Levels are Responsive to cloning AND expression Rhamnose Concentrations Between 0.001% &  Eliminate “leaky” expression with glucose 0.2% 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 Control the Timing of Protein Expression Alter Glucose Conc. for Toxic Target Autoinduction

Early Autoinduction Late Autoinduction Hours 0 6 8 10 24 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 Poll QuestionQuestion #3

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 • 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 • Cleave SUMO tag using SUMO Express Protease • Easily remove protease with Ni2+ column purification

Select Your Promoter • 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 SUMO 6xHis target protein by IMAC Tag Target Protein (Ni2+ column).

SUMO 6xHis 2) Add SUMO Express + Protease Protease (with 6xHis tag) and incubate. SUMO SUMO 6xHis 6xHis 3) Remove 6xHis-SUMO tag Tag + Target Protein + Protease and SUMO Express Protease by subtractive 2+ IMAC (Ni column). + Subtractive IMAC

4) Purified, native protein is recovered from the Target Protein column flow-through. 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 Methods are problematic. Requires high degree of skill. re-fold protein. Optimize protein induction parameters, export. Time-consuming, may not work.

Use optimized system with fusion partners to Screen multiple fusion tags simultaneously with tunable enhance solubility and expression levels. 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 Stabilize mRNA and protein with fusion tags. or mRNA degradation

Help overcome codon bias at the 5’ end of Low expression due to codon sequence with N-terminal fusion tags. Codon bias bias at 5’ end often stalls translation.

Tightly control expression levels with Toxicity 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:

 Improve expression with tunable promoter ONLY kit with a panel  Produce native protein with SelecTEV™-cleavable tags of fusion tags  Purify native protein from 6xHis-SelecTEV™ Protease Expression & Solubility Screening Workflow “Gene to Protein” in as Few as 4 Days

Day 1 Day 2 Day 3 Day 4

Amplify your gene. Purify Design Evaluate protein (Ni2+) one set of Clone your PCR product Start cultures target and remove Soluble, PCR into all seven Expresso® for protein fusion tags native primers to fusion vectors plus the expression expression with protein. amplify control vector. (same strain). and SelecTEV™ your gene. solubility. Transform E. coli. Protease. 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

AFV slyD tsf SUMO Bla MBP GST Control T S I T S I T S I T S I T S I T S I T S I T S I

* * * * * * * *

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. 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 Comparison of OverExpress with BL21(DE3) BL21 (DE3) for toxic proteins (membrane proteins, cytoplasmic proteins, nucleases)  Proven success with over 350 referenced publications  Reduced expression from T7 promoters  Strains differ in tolerance for different proteins

 Also available as pLysS for Dumon-Seignovert, et al. , (2004). Protein Expression and Purification 37, 203-206. tighter expression control

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 SDS- PAGE 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 Express an Express a Improve or Express high levels insoluble toxic optimize membrane of protein protein protein expression proteins from conditions T7 promoter Expresso® T7 Cloning and Expression System √ Expresso® T7 SUMO Cloning and Expression System √ √ Expresso Rhamnose Cloning and Expression System √ Expresso Rhamnose SUMO Cloning and Expression System √ √ Expresso Solubility and Expression Screening System √ √ √ √ OverExpress™ C41(DE3) and C43(DE3) competent cells √ 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

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