- Making the right connections - Disulfide Bond Formation in the Cytoplasm of E. coli

1. C’ N’ ? - Making the right connections - Disulfide Bond Formation in the Cytoplasm of E. coli Mehmet Berkmen

2. Protein folding is a complex problem For correct folding, proteins may require - secondary and tertiary structure - oligimerization - co-factor association - processing - covalent modifications - lipidation - glycosilation - methylation - disulfide bond formation Temporal and spatial coordination of these events is necessary

3. + cytoplasmic isomerase/ chaperone Construction of a novel E. coli strain capable of correctly oxidizing proteins oxidizing periplasm oxidizing and reducing cytoplasm SHUFFLE wt+

4. Cys CH2 S S CH2 Cys Disulfide Bond Formation Cys CH2 SH Reduction + 2H+ + 2e- Oxidation SH CH2 Cys

5. Prokaryotic Eukaryotic disulfide bond formation cytoplasm Eukaryotic Endoplasmic Reticulum Gram negative bacterial periplasm dsbA dsbC PDI PDI • disulfide bonds are important for protein folding and stability • found in secreted extracytoplasmic proteins • antibodies, hormones, proteases, toxins, cellular signals

6. %85 %15 The periplasm of E. coli might not be optimum for production of multi disulfide bonded proteins • MBP fusion constructs yield 10X more in the cytoplasm compared to the periplasm • only 15% of the 1550 exported proteins in E.coli have 2 or more cysteines

7. Expression of complex disulfide bonded proteins in E. coli require the isomerase DsbC Increasing disulfide bond complexity Increasing dependence on DsbC

8. Design and Construction of SHuffle trx/grx dsbC trx/grx dsbC Origami = oxidizing cytoplasm cytoplasm periplasm OR Endoplasmic Reticulum SHuffle = Origami + cytoplasmic DsbC

9. Trx Trx SH SH SH SH SH SH SH SH Glutathione (gshA, gshB) S S S S S S S S DsbC DsbC Designing SHuffle wt+ S S S S DsbA DsbA oxidative periplasm Grx Grx reducing cytoplasm Glutaredoxins Thioredoxins Grx1 Grx2 Grx3 (grxA, grxB, grxC) Trx1 Trx2 (trxA, trxC) Thioredoxin Reductase Glutathione Reductase (gor) (trxB) NADPH

10. Trx Trx SH SH SH SH SH SH SH SH S S S S S S S S DsbC DsbC Thioredoxins Trx1 Trx2 (trxA, trxC) FÅ113 aka ORIGAMITM S S S S DsbA DsbA Grx Grx D trxB D gor ahpC* Glutaredoxins Grx1 Grx2 Grx3 (grxA, grxB, grxC) Peroxidase (ahpC*) Peroxidase Reductase (ahpF) NADH

11. Trx Trx SH SH SH SH SH SH SH SH S S S S S S S S S S S S S S S S DsbC DsbC DsbC DsbC Thioredoxins Trx1 Trx2 (trxA, trxC) SHuffle DsbA DsbA Grx Grx D trxB D gor ahpC* + chromosomal rrnBP1-cytoDsbC Glutaredoxins Grx1 Grx2 Grx3 (grxA, grxB, grxC) Peroxidase (ahpC*) Peroxidase Reductase (ahpF) NADH

12. DsbC is also a general chaperone and might help fold proteins that do not require disulfide bonds DsbC % GAPDH Reactivation D-glyceraldehyde-3-phosphate dehydrogenase (no S-S) PDI monomeric DsbC BSA Protein / GAPDH Chen, Song, Zhang, Wang, Cui, Wang JBC (1999) 28:19601-5

13. SHuffle • What is it good for? Unlike wild type E. coli, SHuffle is an E. coli strain that can promote correct disulfide bond formation in the cytoplasm thus…. Proteins which require disulfide bonds for their folding can now be expressed in the cytoplasm of SHuffle • Who would use it? Scientist/Industry trying to make non-cytoplasmic proteins e.g. antibodies, hormones, proteases, toxins, signaling peptides

14. SHuffle vs Origami • usingTissue plasminogen activator (tPA) • serine protease involved in the fibrinolytic system that dissolves blood clots thrombolytic agent (clot-busting drug) • in 1996 FDA approved the use of tPA to treat ischemic stroke in the first three hours after the start of symptoms tPA structure – six disulfide bonds

15. Tissue plasminogen Activator (9 S-S) vtPA cytoplasm trxB gor ahpC* (FÅ113) cDsbA cDsbC TrxA CGPC wt+ TrxA CPYC grx Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm compartment periplasm genotype wt+ co-expressed foldase DsbA DsbC Bessette PH, Åslund F, Beckwith J, Georgiou G. PNAS 1999

16. Production of correctly folded Fab antibody fragment in the cytoplasm of E. coli trxB gorahpC* (FÅ113) via the coexpression of molecular chaperones * * Levy R, Weiss R, Chen G, Iverson BL, Georgiou G. Protein Expr Purif. 2001 (2):338-47.

17. Tissue plasminogen Activator (9 S-S) vtPA trxB gor ahpC* + cytoplasmic DsbC

18. WT WT cut cut uncut uncut • 1,3 Galactosidase with two cysteines from Xanthomonas manihotis Extract titers Periplasmic a-Gal 1,700 U/gr cells Shuffle Cytoplasmic a-Gal 12,500 U/gr cells

20. Growth curves of various Shuffle strains at 30º C in RICH media Shuffle E. coli K12 Shuffle Express E. coli B

21. SHuffle is more robust when using the T7 expression system - IPTG + IPTG Shuffle + IPTG Origami (DE3)

22. Use of Serratia marcescens extracellular nuclease (2 S-S) for Shuffle QC pMBP-nucA pMBP DHB4 wt+ Shuffle

23. E. coli K12 E. coli B NEB 10b SHuffle SHuffle Express T7 T7 LysY T7 T7 LysY C3025 C3026 C3027 C3028 C3029 C3030 C3019 Available for sale!!!!

24. SHuffle Shuffle™ lacks the cytoplasmic reductases making its cytoplasm amicable to the formation of disulfide bonds. Further enhancement in disulfide bond fidelity was engineered with the chromosomal constitutive expression of the disulfide bond isomerase, DsbC.-        Shuffle™ is constructed in two E. coli strain backgrounds. E. coli K12 (Shuffle™) and E. coli B (Shuffle™ Express) for enhanced protein expression.-        We have also engineered into the chromosome the T7 RNA polymerase enabling the expression of proteins from the T7 promoter. (Shuffle™ T7 and Shuffle™ T7 Express)-        For the expression of toxic proteins from the T7 promoter, we have introduced an episome harboring mutant T7 RNA polymerase inhibitor LysY lacking its lysozyme activity (Shuffle™ T7 lysY and Shuffle™ T7 lysY Express)Click here for detailed information about NEB’s Shuffle™ strains.We are currently offering 6 versions of Shuffle™.  All are available in one size: 6 x 0.05 ml for $60 US.E. coliK12 strains;Shuffle™……………………..Cat# C3025HShuffle™ T7………………….Cat# C3026HShuffle™ T7 lysY……………..Cat# C3027HE. coliB strains;Shuffle™ Express……………..Cat# C3028HShuffle™ Express T7………….Cat# C3029HShuffle™ Express T7 lysY. …....Cat# C3030HThe T7 versions allow for the expression of proteins from the T7 promoter. LysY is an inhibitor of the T7 RNAP and allow for the expression of toxic proteins.

25. THE MARKET: Examples of publications (>200) using Origami « « « «

26. « « « THE MARKET: Publications using Origami «

27. THE COMPETITION: - NovagenCompetent Cells + pRARE tRNA genes (CamR) 70626: Origami 70630: Origami (DE3) 70629: Origami (DE3)pLacI 70618: Origami (DE3)pLysS (Glycerol Stock) 71344: Origami 2 71345: Origami 2 (DE3) 71347: Origami 2 (DE3)_pLacI 71346: Origami 2 (DE3)pLysS 70836: Origami B 70837: Origami B (DE3) 70838: Origami B (DE3)pLacI 70839: Origami B (DE3)pLysS 71054: Rosetta-gami 71055: Rosetta-gami (DE3) 71056: Rosetta-gami (DE3)pLacI 71057: Rosetta-gami (DE3)pLysS 71350: Rosetta-gami 2 71351: Rosetta-gami 2 (DE3) 71353: Rosetta-gami 2 (DE3)pLacI 71352: Rosetta-gami 2 (DE3)pLysS 71135: Rosetta-gami B 71136: Rosetta-gami B (DE3) 71138: Rosetta-gami B (DE3)pLacI 71137: Rosetta-gami B (DE3)pLysS Origami genotype: D(ara-leu)7697 DlacX74 DphoA PvuII phoR araD139 ahpC galE galK rpsL F′ [lac+ lacIq pro] gor522::Tn10 trxB - (KanR, StrR, TetR) Origami 2 genotype: D(ara-leu)7697 DlacX74 DphoA PvuII phoR araD139 ahpC galE galK rpsL F′ [lac+ lacIq pro] gor522::Tn10 trxB - (StrR, TetR)

28. Conclusion SHuffle can correctly fold proteins which require disulfide bonds in the cytoplasm e.g. - extracytoplasmic proteins (periplasmic, ER, Golgi, vacuoles) - extracellular secreted proteins - hormones, proteases, receptors, antibodies, toxins SHuffle is better than Origami for - complex disulfide bonded proteins - T7 over expression SHuffle is just as good as Origami for - simple disulfide bonded proteins Recommended expression conditions: SHuffle express, 30º C, Rich media

29. Work in progress… • - extensive characterization of Shuffle • expression conditions (Tº, induction, media) • more substrates • active soluble protein yields