Chaperone Co-expression Strategies for Soluble Protein Production in E. coli

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1 Chaperone Co-expression Strategies for Soluble Protein Production in E. coli Bo Wu, Ph.D.

2 Table of Contents E. Coli Expression Overview Inclusion Bodies & Refolding Expression Test & Optimization Fusion Partners Chaperones Advantages of GenScript Protein Services 2

3 Expression Systems Bacteria Insect Yeast Mammalian Cell Free E. coli 1. Work horse 2. Well established 3. High expression 4. Simple genetics Sf9, Sf21, S2, High-5 1. PTMs 2. Soluble proteins 3. High expressers S. cerevisieae P. pastoris 1. PTMs 2. Soluble proteins 3. High expresser CHO, HEK, COS 1. PTMs 2. Soluble proteins 3. Low expresser 4. Expensive In vitro 1. Expensive 2. Not reproducible 3. Scalability issues 5. Easy scale up 6. Speed 7. Costs 8. Equipment Before Embarking on a Protein Expression Project 3

4 E. coli Is the Top Choice 100% PDB entries reflects dominance of E. coli expression 92% 80% 60% 40% 20% 0% 3.8% 2.06% 1.03% 0.29% E.coli Insect Yeast Mammalian Cell Free Number of entries in the PDB by expression system as a percentage of total number of chains with an identifiable expression system, as of April 15, All values are approximate. Reference - E.coli expression 4

5 Introduction to E. coli Expression Escherichia coli [E. coli] is work horse Variety of host species available Successful implementation of bacterial protein production project dependent on several factors Recommendations for optimizing recombinant protein production in E. coli We describe learnings from our collective experience GenScript Production Team has shipped over 3,000 batches of recombinant proteins Introduction 5

6 E. coli Expression Why & Why Not? Advantages Simple genetics Easy to manipulate Inexpensive to culture Easy to Scale up Fast expression High yields Disadvantages Lack of PTM Codon usage Inclusion bodies Low yield of many eukaryotic proteins Poor secretion High MW proteins difficult No/Low Expression Insoluble Expression E.coli expression 6

7 Table of Contents E. Coli Expression Overview Inclusion Bodies & Refolding Expression Test & Optimization Fusion Partners Chaperones Advantages of GenScript Protein Services 7

8 Protein Expression as Inclusion Bodies What is an inclusion body? When E.coli is transformed to manufacture large amounts of recombinant protein, the protein sometimes forms dense aggregates of insoluble misfolded proteins, known as inclusion bodies. Benefit allow high protein concentrations protect sensitive proteins from proteolytic (enzymatic) degradation protect the cell from any toxic proteins Challenge to solubilise and refold this protein into its correct active form 8

9 Protein Refolding Challenges All the information necessary for folding the peptide chain into its native structure is contained in the primary amino acid sequence of the peptide. There are vastly too many different possible conformations for a protein to fold by a random search. A new view of protein folding suggested that there is no single route, but a large ensemble of structures follow a many dimensional funnel to its native structure. Refolding conditions must be optimized for each individual protein. Important variables are: buffer type ph ionic strength Additives, often in combination (glycerol, redox reagents, saccharides, amino acids, metal ion, detergents, chaperones) 9

10 GenScript s FoldArt TM Technology Overview Evaluation of target proteins' biochemical and biophysical properties Refolding optimizations Selection of particular refolding strategy based on protein's sequence and the structural properties. Buffer screening: Solutions for the inclusion body will be diluted to 20 different refolding buffers to determine which parameters affect the refolding results. Important buffer variables are: buffer type ph ionic strength Additives, often in combination (glycerol, redox reagents, saccharides, amino acids, metal ion, detergents) Denaturant removal Techniques: dilution, dialysis, diafiltration, and chromatography (ion exchange, size exclusion, and affinity) Validation Refolding results will be validated by SDS-PAGE, HPLC and/or functional assay. 10

11 Case Study: Protein Refolding Human interleukin 5: disulfide-bond linked homodimer as active form Dimer 11

12 Table of Contents E. Coli Expression Overview Inclusion Bodies & Refolding Expression Test & Optimization Fusion Partners Chaperones Advantages of GenScript Protein Services 12

13 PROTential TM - Expression Evaluation & Optimization Eliminate the guesswork from your protein production work Evaluate whether your target protein expresses in your chosen system Identify the best expression system for your target protein PROTential TM Standard packages Before scale-up protein production, to avoid waste on your time & valuable resources Optimize your protein expression PROTential TM Silver & Gold packages When challenges arise the most efficient & cost-effective way One stop service at GenScript: gene synthesis Subcloning PROTential TM Scale up protein production GenScript s Solution for Expression Optimization 13

14 PROTential TM - Portfolios 14

15 Case Study- Protein Expression Optimization Challenges: Pilot purification (final yield 1mg/L )- ~ 28kDa protein; The protein can be only purified from the soluble part Large amount of protein with large scale fermentation (1000L) and purification is needed. Strategies: 1. Expression improvement: a. Promoter optimization b. Strain optimization c. ph optimization d. Temperature and induction optimization e. Inoculated quantity optimization 2. Recovery rate improvement during purification - Purification condition optimization 15

16 Case Study- Protein Expression Optimization 12X as much protein expression yield as original 1 mg/l starting protocol 2 mg/l T7 promoter/induction condition optimization 5 mg/l phoa promoter/induction condition optimization 6 mg/l growth condition optimization (ph) 12 mg/l seeding density optimization 16

17 Factors Critical to Solubility Inducer Growth Media I. Codon Optimization II. Strain SOLUBLE PROTEIN III. Temperature IV. Fusion Partner Induction time Chaperones & Foldases Vectors & Promoters Factors Critical to Solubility 17

18 Table of Contents E. Coli Expression Overview Inclusion Bodies & Refolding Expression Test & Optimization Fusion Partners Chaperones Advantages of GenScript Protein Services 18

19 Fusion Partner/Affinity Tag Tag ~MW [kda] Pros Cons 6His 1 Universal, small, works with native and denaturing conditions Usually requires more than 1-step elution. Not entirely innocuous CBP 4 High specificity Purity differs greatly Flag 1 GST 26 High purity Introduces Enterokinase Elution easy, functions as solubilizing tag MBP 40 Solubility enhancer Strep 1 High specificity, Mild elution conditions Expensive and elution can be a problem occasionally Dimerizing nature Leaches occasionally Need longer contact times, Relatively large tag Need longer contact times NusA 55 Solubility enhancer Very large tag Trx 12 Solubility enhancer Does not work well with larger MW target proteins Factors Critical to Solubility 19

20 Introduction to MBP Tag Maltose binding protein (MBP) is a solubility enhancing tag. Secreted E. coli protein, effective for affinity purification Functions: Uptake, breakdown and transport maltodextrin Stabilize and protect its downstream passenger protein from proteolytic degradation during and after protein synthesis Provide reliable contexts for efficient translation initiation May act as a chaperone to stabilize otherwise insoluble passenger protein Common construct design strategy 20

21 Introduction to NusA Tag NusA is a large N-utilizing substance A transcription antitermination factor. Usually in combination with His-tag Functions: Promote hairpin folding Anti-termintation and termination PDB ID: 2ATW 21

22 Comparable Effect of MBP and NusA target tag simultaneous tag removal protein MBP NusA MBP NusA A B C D E (++++ > 75soluble; % soluble; + < 25% soluble; - insoluble) The ability of MBP and NusA to promote the proper folding of their fusion partners is similar. Functional assay showed MBP and NusA had comparable enzymatic activity. The underlying mechanism of solubility enhancement seems to be similar between MBP & NusA. (Protein Expression and Purification 45 (2006) ) 22

23 Introduction to Trx Tag Thioredoxin (Trx) is a thermostable intracellular E. coli protein First fusion tag used to increase solubility of a target protein More effective when placed on the N-terminal The Trx domain presents in a lot of chaperones PDB ID: 1KEB 23

24 Case Study of a Trx Fusion Protein kda 94 M Target protein after digestion was purified with Nickel column; fractions were analyzed by SDS-PAGE followed by Coomassie Brilliant blue staining Lane M: Protein marker Lane 1: Fusion protein Lane 2: Digestion mixture Lane 3-5: Flow through 24

25 Table of Contents E. Coli Expression Overview Inclusion Bodies & Refolding Expression Test & Optimization Fusion Partners Chaperones Advantages of GenScript Protein Services 25

26 Use of Co-expression Strategy Commonly used chaperones: PDI GroES/L TF Novel chaperones: Skp FkpA Lysis partner: VanX 26

27 Solubility Enhancement by Chaperones OD no-coexpression Skp coexpression FkpA coexpression Co-expression of FkpA increased the soluble expression of a scfv fragment over 10 times determined by antigen-binding ELISA 27

28 Effect of DsbC and Erv1p MBP-tag with BL21 His-tag with Origami Fold activity MBP-tag with BL21 Fold activity His-tag with Origami 0 no-coexpressioncoexpression DsbC/Erv1p 0 no-coexpression pre-expression DsbC/Erv1p Both co-expression and pre-expression of DsbC and Erv1p increased soluble expression of a target protein, thus increased the activity of the target protein. 28

29 Lysis Partner VanX no VanX with VanX intracellular media When VanX is present, protein is released to the media instead of staying intracellular. 29

30 Table of Contents E. Coli Expression Overview Inclusion Bodies & Refolding Fusion Partners Chaperones Case Studies of Chaperones Advantages of GenScript Protein Services 30

31 Advantages of GenScript Protein Services Core in-house technologies for expression optimization & production efficiency. OptimumGene TM expression system specific codon optimization BacPower TM increase bacterial soluble expression FoldArt TM ensure appropriate protein refolding YeastHigh TM high copy-number gene selection technique BacuVance TM for protein secretion from baculovirus-infected insect cells MamPower TM technology licensed from NRC for rapid recombinant protein production with high yield DoubleTag strategy for big protein isolation 31

32 Advantages of GenScript Protein Services One-stop service from sequence to purified proteins with large capacity. 3 mg purified soluble protein from $2,200 Subcloning Transformation Expression Refolding Bacteria Yeast Baculovirus/insect cells Mammalian Bacterial fermentation up to 1,000 L Yeast fermentation up to 500 L Baculovirus/insect cell production up to grams Mammalian cell production up to grams Mammalian protein expression services Stable cell line development & protein production 32

33 Advantages of GenScript Protein Services Flexible production scales Fast turn-around time (from sequence to purified protein in as little as 4 weeks) Capacity: Bacteria Yeast Baculovirus Mammalian 1,000 L 500 L 100 L 500 L Fermentor Fermentor Wave TM Mixer Wave TM Mixer Sartorious bioreactor Hyclone SUB bioreactor 33

34 GenScript Protein Standard Services From sequence to purified protein - gene synthesis included! Expression System Deliverables Timeline Price BacPower TM 3-10 mg purified protein guaranteed 6-8 weeks Staring from $2,200 InsectPower TM 1mg purified protein guaranteed 8-10 weeks Staring from $3,950 MamPower TM 1-10mg purified recombinant protein or 5-500mg purified antibody guaranteed 8-12 weeks Staring from $2,999 YeastHigh TM Customizable production up to 2000L 8-10 weeks Quote 34

35 GenScript s Experience in Protein Expression & Purification GenScript has delivered over 5,000 proteins in four expression systems. Statistics showed 95% success rate for all protein projects. 35

36 About GenScript Gene Cell Line Peptide Discovery Biology GenScript Products & Services Protein Antibody 36

37 Thank you for your participation We wish you all success in your Research Register for other webinars in the GenScript Webinar May 20, 2015/ 9:00 am EDT Analyzing antibody sequence for recombinant antibody expression Hangxing Yu, Ph.D June 3, 2015/ 11:00 am EST Expression vectors: how to choose, or customize vectors for gene & protein expression Rachel Speer, Ph.D. Promo Code: BacWebinar % off any BacPower TM Guaranteed Packages - Valid till 8/15/2015 Conclusion 37