Strategies for Improving Soluble Protein Production in E. coli
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- Muriel Johns
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1 Strategies for Improving Soluble Protein Production in E. coli
2 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
3 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
4 Applications for Recombinant Proteins Crystal structure determination Enzyme Classification Industrial enzymes for consumer products Drug discovery doi: /journal.pone g004 Biotherapeutics Research tools Human kinases (>500)
5 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 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
6 General Protein Expression and Purification Workflow Cloning Through in vitro Analysis Cloning Expression Purification Analysis 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 with toxic targets Low expression levels Low solubility (inclusion bodies) Protein degradation or truncation Poor cell viability with toxic targets Need to transfer vector from cloning to expression strain Non-optimized purification strategies Occluded purification tags Contaminating proteins Ineffective cell lysis method Poor salt and detergent choices False solubility Inactive protein Insufficient protein yield Insufficient protein purity Endotoxin contamination Incorrect posttranslational modifications
7 Protein Expression in E. coli Workflow from Gene to Protein Promoter Tag Replication Origin Selection Marker Clone Target Gene 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 Promoter Tag Replication Origin Target Gene Selection Marker Transform E. coli E. coli Express Protein Target Gene Evaluate
8 Choose an Expression Vector Vector Determines Downstream Strategy 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 Promoter Promoter Tag Replication Origin Tag Target Gene Replication Origin Selection Marker Clone Target Gene Selection Marker Transform E. coli Express Protein E. coli Selection Marker Replication Origin Promoter Tag Target Gene Evaluate
9 Insert the Target Gene Many Cloning Technologies are Available PCR-amplicon Cloning Workflow Promoter Tag Replication Origin Selection Marker Clone Target Gene Other Methods: Restriction enzyme-based ( cut and paste ) LIC (Ligation Independent Cloning) In-Fusion TOPO Recombinational (Gateway ) Gibson Cloning (SGI-DNA, NEBuilder ) Promoter Tag Replication Origin Target Gene Selection Marker Transform E. coli E. coli Express Protein Target Gene Estimated preparation time: ~10 hours Evaluate
10 Transfer the Clone into E. coli Different Methods Address Different Needs 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 Promoter Promoter Tag Replication Origin Tag Target Gene Replication Origin Selection Marker Clone Target Gene Selection Marker Transform E. coli Express Protein Target Gene E. coli Evaluate
11 Induce Expression and Produce Protein Successful Results Depend on this Strategy Promoter Tag Replication Origin Selection Marker 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 Promoter Tag Target Gene Replication Origin Clone Target Gene Selection Marker Transform E. coli Express Protein Target Gene E. coli Evaluate
12 Protein Expression at the Molecular Level Many Potential Challenges Exist Improper folding Insoluble Protein Transmission electron microscopy Origins of error: Low trna availability mrna secondary structure Translation rate too slow or too fast Expression level too high Toxicity Coupled transcription/translation Degraded Protein
13 What Can Go Wrong and Why? Issue Possible Explanations Potential Solutions No or low expression Formation of inclusion bodies Inactive protein Protein may be toxic to cell before or after induction Codon bias Improper folding, low solubility Incorrect disulfide formation Missing post-translational modification Incomplete folding Mutations in cdna Lacking post-translational modifications or cofactors Suppress basal induction of protein- use tight promoters, defined media Use special engineered hosts Use tunable promoters Reduce copy number Direct protein to periplasm 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 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 Direct protein to periplasm Use special host with oxidative environment Use an alternate host Reduce growth temperature, promote disulfide bond formation Confirm plasmid sequence Use reca- strains to maintain stability in cell Use fresh transformants Coexpress transferases, add cofactors Switch to yeast, insect or mammalian cell systems
14 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
15 Question #3 Poll Question What is the native source and type of the protein(s) you are trying to express? Choose all that apply.
16 General Protein Expression and Purification Workflow Cloning Through Analysis Cloning Expression Purification Analysis 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 with toxic targets Low expression levels Low solubility (inclusion bodies) Protein degradation or truncation Poor cell viability with toxic targets Need to transfer vector from cloning to expression strain Non-optimized purification strategies Occluded purification tags Contaminating proteins Ineffective cell lysis method Poor salt and detergent choices False solubility Inactive protein Insufficient protein yield Insufficient protein purity Endotoxin contamination Incorrect posttranslational modifications
17 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 Colony PCR Shows >90% Correct Clones Uses pre-processed, linearized vector Highly efficient
18 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
19 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
20 Expresso Cloning Primer Design Design PCR Primers with Overlapping Sequences Forward primer: 5 CAT CAT CAC CAC CAT CAC nucleotides gene specific sequence Reverse primer: 5 GTG GCG GCC GCT CTA TTA nucleotides reverse compliment gene specific sequence
21 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
22 Poll Question Which challenges are you facing in your protein expression projects? Choose all that apply.
23 General Protein Expression and Purification Workflow Cloning Through Analysis Cloning Expression Purification Analysis 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 with toxic targets Low expression levels Low solubility (inclusion bodies) Protein degradation or truncation Poor cell viability with toxic targets Need to transfer vector from cloning to expression strain Non-optimized purification strategies Occluded purification tags Contaminating proteins Ineffective cell lysis method Poor salt and detergent choices False solubility Inactive protein Insufficient protein yield Insufficient protein purity Endotoxin contamination Incorrect posttranslational modifications
24 Common Protein Expression Challenges and Solutions Key Roadblocks to Soluble, Active protein Degradation Codon Bias Toxicity Inclusion Body Formation
25 How Can the Expresso T7 Cloning and Expression System Help? Toxicity Challenge Lucigen Solutions Control basal expression with HI-Control cells. Low Solubility Enhance solubility with optional SUMO tag. Low Expression Time-Consuming Cloning Produce high levels of protein from inducible T7 promoter. Save time and increase throughput with Expresso cloning.
26 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
27 Reduce Problematic Leaky Expression Clone and Express Challenging Targets with HI-Control Problem: Background expression of T7 RNA Polymerase = Uninduced target gene expression HI-Control Cells Express LacIq Repressor Lac Repressor Decreased colony count and cell viability during cloning and expression LacUV5 Promoter Lac Operator T7 RNA Polymerase Solution: HI-Control BL21(DE3) T7 Promoter Lac Operator Target Gene HI-Control 10G (cloning strain) + IPTG Increased expression of LacIq repressor = Improved control of gene expression Induced Target Gene Expression Less toxicity and more soluble protein T7 RNA Polymerase Lac Operator Target Gene
28 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
29 How Can the Expresso Rhamnose Cloning and Expression System Help? Toxicity Challenge Low Solubility Low Expression Time-Consuming Cloning and Transfer Lucigen Solutions Control basal expression and fine-tune induction level of toxic proteins with tunable rhamnose-inducible promoter. Enhance solubility with optional SUMO tag. 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.
30 Tunable Protein Expression with Virtually No Background Ideal System for Toxic Proteins Fine-Tune Expression Levels Rhamnose-inducible rhap BAD promoter Enhanced solubility Select Optimized Vectors N-or C-terminal 6xHis tag Small: ~2.3kb Kanamycin resistance Transcriptional terminators Optional SUMO tag Express Toxic Proteins Eliminate leaky expression with glucose repression Express proteins in almost any E. coli cell line Transcriptional terminators stabilize clones by preventing toxicity due to transcription into and out of cloned fragments
31 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%
32 Control the Timing of Protein Expression Alter Glucose Conc. for Toxic Target Autoinduction Hours Early Autoinduction Late Autoinduction Late autoinduction: Start autoinduction cultures with more glucose (repressor) to delay protein expression Build up more biomass before induction begins Produce higher protein yields
33 Question #3 Poll Question Which fusion tags have you used to express your protein in E. coli? Choose all that apply.
34 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 Ni 2+ column purification Select Your Promoter Expresso T7 SUMO Cloning and Expression System for high-level production Expresso Rhamnose SUMO System for controlled, tunable production
35 Recover Native Proteins Quickly Simplify Purification and Tag Cleavage 1) Express and purify tagged target protein by IMAC (Ni 2+ column). 6xHis SUMO Tag Target Protein 2) Add SUMO Express Protease (with 6xHis tag) and incubate. 3) Remove 6xHis-SUMO tag and SUMO Express Protease by subtractive IMAC (Ni 2+ column). 4) Purified, native protein is recovered from the column flow-through. SUMO Tag + 6xHis 6xHis + Target Protein + Target Protein SUMO Protease 6xHis + Subtractive IMAC SUMO Protease
36 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.
37 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 Purify protein as inclusion bodies. Denature and re-fold protein. Optimize protein induction parameters, export. Use optimized system with fusion partners to enhance solubility and expression levels. Impact Methods are problematic. Requires high degree of skill. Time-consuming, may not work. Screen multiple fusion tags simultaneously with tunable promoter, easy expression protocol and fast cloning method.
38 How Can the Expresso Solubility and Expression Screening System Help? Low Solubility Problem Source Lucigen Solutions Enhance solubility with a panel of fusion tags. Low expression due to protein or mrna degradation Low expression due to codon bias Toxicity Need for multiplex capabilities Stabilize mrna and protein with fusion tags. Help overcome codon bias at the 5 end of sequence with N-terminal fusion tags. Codon bias at 5 end often stalls translation. Tightly control expression levels with rhamnose promoter. Use a high-throughput compatible cloning and screening workflow. Save time by using a single strain for cloning and expression.
39 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
40 Expression & Solubility Screening Workflow Gene to Protein in as Few as 4 Days Day 1 Day 2 Day 3 Day 4 Design one set of PCR primers to amplify your gene. 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). Evaluate target protein expression and solubility. Purify protein (Ni 2+ ) and remove fusion tags with SelecTEV Protease. Soluble, native protein.
41 High Efficiency Speeds Workflows, Enables Automation Clone Into 7 Expresso Vectors with >90% Efficiency 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 24 / 24 Correct Clones by Colony PCR Screen of SOL-LIN28 Target
42 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.
43 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).
44 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 custserv@lucigen.com Custom Solutions
45 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) 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 Comparison of OverExpress with BL21(DE3) Dumon-Seignovert, et al., (2004). Protein Expression and Purification 37, Literature references:
46 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 30 o C or 22 o C 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
47 Summary If You Need To Express high levels of protein Express an insoluble protein Express a toxic protein Improve or optimize expression conditions Express membrane proteins from 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
48 Resources Poster: Nature Methods: Application Notes:
49 Questions? Thank You! Lucigen Tech Support (888) (608) am 5 pm Central Time
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