Optimizing conditions for recombinant soluble protein production in E.coli. Keshav Vasanthavada, MSc., MS May 8 th, 2014

Transcription

1 Optimizing conditions for recombinant soluble protein production in E.coli Keshav Vasanthavada, MSc., MS May 8 th, 2014

2 Presentation overview Introduction Before embarking on a protein expression project E.coli expression GenScript s solution for expression optimization Factors critical to solubility Conclusion 2

3 About GenScript Gene Cell Line Peptide Discovery Biology GenScript Products & Services Protein Antibody Introduction 3

4 Historical perspective One Gene One Enzyme Hypothesis (Beadle & Tatum) 1953 elucidation of the helical nature of DNA (Franklin & Wilkins) Solving the 3-D structure of DNA (Watson & Crick) First recombinant protein (somatostatin) made in E. coli 1977 DNA sequencing (Maxam & Gilbert) 1980 PCR was developed (Kary Mullis) Full length synthetic Proteins? Protein Amplification Technology? Central Dogma (Francis Crick) 1970 Discovery of Restriction Enzymes (Smith & Nathans) 1973 Birth of Recombinant DNA Technology (Cohen & Boyer) 1987 Refolding of Inclusion bodies from E. coli (Buchner, Kelley, Rudolph) 1992 Baculovirus Expession Vector System (Summers, 1983; King & Posse, 1992) Mammalian cell expression (Hauser 1988; Page 1991; Geisse 1996) 1993 Affinity purification of His- & GST-tagged proteins (Hochuli 1988; Jones 1993) Introduction 4

5 Introduction to topic Recombinant protein production (RPP) is essential to functional characterization and structure determination Variety of hosts available for RPP Scientists faced with overwhelming choices Escherichia coli [E. coli] is work horse Successful implementation of bacterial protein production project dependent on several factors Observations & 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 Common questions Activity My Protein My Own My Precious But me why needs it? How is me making it? Before embarking on a protein expression project 6

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

8 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 8

9 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 Low/No Expression Insoluble Expression E.coli expression 9

10 What is the problem? what are the solutions? E.coli Refold No Target Engineering Temperature Strains Media Buffers Chaperones ~24,000 Targets NESG Complete Inclusion Bodies Optimize 43% 57% Soluble Insoluble Engineer Target Truncate Mutate Fusion Partner E.coli expression 10

11 Goal of a protein production campaign Variables Desirables Generation of high yields of soluble protein in a cost-effective and expeditious manner is the single most important determinant of a successful protein production campaign E.coli expression 11

12 Protein expression campaign workflow Project initiation Literature Review Target Analysis & Engineering Host Consideration Cloning scheme/gene synthesis Small scale expression optimization Scale up and Purification What has been done already? Homologs, truncations, mutations? Full-length or fragment? Tag or tag-less? Which tag? N or C-terminus? E.coli expression 12

13 Flow chart of a bacterial expression project WITHOUT target engineering No Discard Re-evaluate Optimize [PROTential TM ] Codon optimization Promoters Vectors Temperature & Culture Strains Media Buffers Chaperone co-expression Optimize [PROTential TM ] WITH target engineering Target Protein Expression in E. coli Insoluble Refold [FoldArt TM ] Truncations Mutants Fusion partners Scale up & Purify Soluble Archive Good idea to run small scale expression and test purification E.coli expression 13

14 PROTential TM - expression evaluation & optimization Eliminate the guesswork from your Protein production work What is PROTential TM? PROTential TM is a new protein expression, evaluation and optimization service offered by GenScript. What are the applications of PROTential TM? Evaluate whether your target protein expresses in your chosen system. Identify the best expression system for your target protein. Identify the construct and conditions that give you the most robust soluble expression of your target protein. Evaluate before scale-up protein production, to avoid wasting your time & resources One stop service at GenScript: gene synthesis Subcloning PROTential TM Scale up protein production GenScript s solution for expression optimization 14

15 PROTential TM portfolio PROTential TM Standard Package (starting from $280) Available for 3 expression systems: bacterial, insect & mammalian Protein expression evaluation & solubility test PROTential TM Silver Package (starting from $1440) Currently for bacterial expression system only Test 8 conditions by combining temperature, media components & inducer concentration. Allow to identify the best expression condition with your chosen vector & bacterial strain. PROTential TM Gold Package (starting from $4800) Currently for bacterial expression system only Test 48 conditions, by combining parameters including not only those 3 covered in silver package, but also promoter, host strain, and fusion partner. The most robust and high throughput protein expression & solubility optimization matrix. Add-on item: 1L bacterial expression with 1 step purification at $600/condition (except for Flag-tagged protein, the cost is $800/condition). GenScript s solution for expression optimization 15

16 PROTential TM standard packages Expression system Price Timeline Deliverables Bacterial Starting from $ weeks Baculovirus/ insect Starting from $ weeks Mammalian Starting from $ weeks SDS & WB result Expression data report All 3 systems Starting from $ weeks Each package represents one gene, one vector and one expression system. These standard packages offer expression and solubility test but no optimization GenScript s solution for expression optimization 16

17 Options for protein tag, growth media & vectors E. Coli Protein Tag His (default) Mammalian Protein Tag His (default) Insect Protein Tag His (default) Gene synthesis customers have listed options with the standard PROTential TM packages. GST Trx MBP SUMO Fc Flag V5 HA Fc Flag GST Strep II If a modification or tag is not on this list, PROTential TM silver or gold package is required in order to accommodate such request. Flag C-Myc MBP Growth Media GST V5 LB (default) HA TB C-Myc 2xYT Vectors pet system pgex system pcold system GenScript s solution for expression optimization 17

18 Other optimization parameters (Gold package only) Promoters T5, T7, trc, tac, CSPA, AraBAD, Lac, Lac UV5, Trp, pl, T7-lac, T5-lac, Lac-ara1, Ltet0-1, PhoA, Psyn, reca, prou, cst-1, teta, cada, nar, T3-lac, T4 gene 32, nprm-lac, lpp, lpp-lac, VHb, or others. Bacterial strains BL21, BL21(DE3), BL21*(DE3), BL21(DE3) plyss (E), BL21-SI, BL21-AI, BL21trxB, BL21 CodonPlus, Origami, Origami B, Origami B plyss, C41(DE3), C43 (DE3), Lemo21(DE3), Turner, Tuner plyss, AE, Rosetta, Rosetta plyss, Rosetta-gami-pLysS, Shuffle, DH5a, TOP10, JM109, TG1, Stbl2, XL10-Gold, AD494, HMS174, NovaBlue(DE3), BLR, HB2151, W3110 or others. Induction conditions Chosen based on our expertise; Can accommodate customer s specific request. Medium components Chosen based on our expertise; can accommodate customer s specific request. Temperature 6C, 10C, 15C, 16C, 20C, 25C, 30C, 37C, or as specified by customer. GenScript s solution for expression optimization 18

19 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 19

20 I. OptimumGene TM codon optimization GenScript has optimized over 50,000 sequences in all major expression systems. The expression level of target proteins can be significantly improved by our OptimumGene TM Codon Optimization Technology. Factors critical to solubility 20

21 Case study: codon optimization Fig. 1: Expression Result of Protein α after Codon Optimization. The expression level of Protein α using GenScript s OptimumGene TM Codon Optimization is 3 times more than that of competitor s. Fig. 2: Expression Result of Protein β after Codon Optimization. The expression level of Protein β using GenScript s OptimumGene TM Codon Optimization is 13 times more than that of competitor s. Factors critical to solubility 21

22 II. Strain # Expression Strain Rationale 1 BL21(DE3) Work-horse of recombinant protein expression. Lamba DE3 lysogen. Basal exp high. Non-toxic preferred 2 BL21(DE3) plyss T7 lysozyme to repress basal level expression 3 BL21(DE3) plyse T7 lysozyme to repress basal level expression. Higher level of repression 4 BL21 Star (DE3) RNaseE (rne131) mutant. Reduced mrna degradation. Higher stability of transcripts 5 BL21 Star (DE3) plyss As above, coupled with higher level of repression 6 BL21 (DE3) Codon Plus RIPL Contain extra copies of amino acids R,I,P,L trna genes. Supplements for codon deficiencies and bias 7 Origami 2(DE3) placi trxb and gor reductase mutants. Enhance disulfide bonds in cytoplasm. Rare trna genes also 8 Origami 2(DE3) plyss trxb and gor reductase mutants. Enhance disulfide bonds in cytoplasm. Lower basal repression 9 Origami B(DE3) placi Enhance disulfide bonds in cytoplasm and control IPTG entry into cells 10 Origami B(DE3) plyss As above, coupled with higher level of repression 11 OverExpress C41(DE3)pLysS Uncharacterized mutation that allows higher production of "difficult" proteins 12 OverExpress C43(DE3) plyss Uncharacterized mutation that allows higher production of "difficult" proteins 13 BL21-Gold (DE3)pLysS Lon and OmpT protease deficient for greater recombinant protein stability. Higher plasmid stability 14 SHuffle T7 Express lysy Enhances disulfide bond formation in cytoplasm, DsbC promotes correction of misoxidized proteins 15 Arctic Express Co-expression with chaperonins Cpn60 & Cpn10 for increased yield of soluble proteins 16 Rosetta 2(DE3)pLysS trna genes for 7 rare amino acids plus reduced basal expression 17 Rosetta-gami B(DE3)pLysS Above plus increased disulfide bond formation in cytoplasm plus reduced basal expression 18 Tuner (DE3)pLacI Lac permease lacy mutant allows uniform entry of IPTG into cells, enhances solubility 19 Tuner (DE3)pLysS Above function plus reduced basal expression 20 Lemo21(DE3) Tunable expression by varying lysozyme concentration, regulated by addition of Rhamnose Factors critical to solubility 22

23 Case study: Impact of E.coli strains on soluble expression 0.5mM IPTG induced/15c Overnight, Ni-IMAC purified Lemo21 (DE3) with varying concentrations of Rhamnose 30kDa A M M mM IPTG induced/15c Overnight, Ni-IMAC purified Lemo21 (DE3) with varying concentrations of Rhamnose 20kDa B M M STRAIN MAKES A DIFFERENCE Factors critical to solubility 23

24 III. Temperature: case study Lower temperatures result in more soluble protein 15C RT 37C 65kDa WB Total Soluble TEMPERATURE MAKES A DIFFERENCE Factors critical to solubility 24

25 IV. 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 Expensive and elution can be a problem occasionally Dimerizing nature Leaches occasionally MBP 40 Solubility enhancer Need longer contact times, Relatively large tag Strep 1 High specificity, Mild elution conditions 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 25

26 Case study: Importance of fusion partner kDa C 15kDa D FUSION PARTNERS MAKE A DIFFERENCE Factors critical to solubility 26

27 Case study: Duration of induction Detectable recombinant protein expression begins as early as minutes Minutes 20kDa E kDa F Longer need not always be better Longer can occasionally be bad kDa G 1. Protein D [0.1mM IPTG, 15C, 3 hours] 2. Protein D [0.1mM IPTG, 15C, Overnight] 3. Protein D [0.1mM IPTG, RT, 3 hours] 4. Protein D [0.1mM IPTG, RT, Overnight] 1. Protein E [0.1mM IPTG, 15C, 3 hours] 2. Protein E [0.1mM IPTG, 15C, Overnight] Factors critical to solubility 27

28 Factors to consider Cloning Strategy LIC or recombination-based cloning strategy for high throughput & parallel construct generation If possible, create expression vectors with N-terminal fusion/solubility partners for easy cloning scheme Expression Express both N- and C-terminally affinity tagged constructs [especially 6His] In many cases at least one version will express If checking for enzyme activity, one version might show higher activity than the other Purification Note that C-terminally affinity tagged constructs allow you to purify full length proteins You could try double-tagging approach by using one affinity tag [e.g., GST, MBP at the N-ter and 6His at the C-ter to solubilize and purify full length protein at the same time Conclusion 28

29 Summary & takeaways No simple, global solution to address insolubility issues in E.coli expression Codon optimization, strain, induction temperature and duration, fusion partners make a difference Small scale expression study should be predictive for early triage of soluble constructs Small scale purification to assess protein behavior and avoid hiccups during scale up Parallel approaches increase the chance of success Suggested approach Combinatorial tagging at the N-terminus [6His + solubilizing tag] Protease Cleavage site for tag removal after 1 st round purification Promoter Protease Linker ATG 6His Solubilizing Tag Protease Tag Target Expression levels, activity, purity, homogeneity, stability are important factors that must be considered Conclusion 29

30 GenScript s experience in protein expression & purification GenScript has delivered over 3,000 proteins in four expression systems with 92% success rate for all protein projects. GenScript has successfully delivered a variety of difficult proteins, including trans-membrane proteins, co-expression proteins, proteases, kinases, cytokines, antibodies and several other proteins to customers worldwide. Conclusion 30

31 Thank you for your participation We wish you all success in your Research Register for other webinars in the GenScript Webinar May 15, 2014/ 2PM EST Avoiding peptide assay failure: hidden problems and solutions - Tiffany Gupton Campolongo, Ph.D. May 21, 2014/ 11:00 am EST Gene variant libraries: design, construction, and research applications - Rachel Speer, Ph.D. May 28, 2014/1:00 pm EST Protein or peptide antigen: choosing the optimal immunogen for antibody production - Jessica Kaplunov, Ph.D. June 5, 2014/ 2:00 pm EST Stem cell culture: choosing optimal conditions for expansion and differentiation - Matthew Riolo, Ph.D. June 11, 2014/ 1:00 pm EST Recombinant protein expression & purification: challenges and solutions - Liyan Pang, Ph.D. June 18, 2014/ 2:00 pm EST Can CRISPR/Cas9 off-target genomic editing be avoided? Ways to improve target specificity - Maxine Chen, Ph.D June 25, 2014/ 2:00 pm EST Building a Synthetic Eukaryotic Genome Sc2.0 - Leslie Mitchell, Ph.D., NYU Langone Medical Center Conclusion 31