1. Protein Expression and Folding Optimization For High-Throughput Proteomics Kate Drahos 9 April 2004

2. It is estimated that 1/3 to 1/2 of prokaryotic proteins cannot be expressed in a soluble form using E. coli expression systems; this estimate most likely increases for eukaryotic proteins (Edwards et al., 2000) Introduction New expression technologies are necessary to provide high quality, labeled, recombinant eukaryotic proteins suitable for structural studies. The fields of structural genomics and protein chemistry rely heavily on recombinant proteins produced in prokaryotic systems. Structural genomics in particular requires large amounts of purified proteins produced in HTP format.

3. Protein Production Pipeline PCR & cloning of ORFs Large-scale production & purification of labeled proteins Construct validation Small-scale expression & solubility screens Structure determination by NMR Structure determination by X-ray crystallography

4. What if…? PCR & cloning of ORFs Construct validation Small-scale expression & solubility screens Unexpressed or Insoluble Proteins!

5. Why don ’ t we get the desired protein product? Requires molecular chaperone for proper folding Requires post-translational modifications Cofactors/protein partners needed for proper folding – up to 1/3 of eukaryotic proteins may be “natively” unfolded until binding their protein partner How can we obtain these proteins as correctly folded molecules?

6. Alternative Expression Constructs 1) Gateway MBP-fusion expression system (Kapust & Waugh 1999) 2) SUMO system (Lifesensors, Inc. Malvern, PA) Attempt to use fusion protein expression systems in place of our standard T7 Multiplex Expression System for a set of 50 eukaryotic target proteins

7. MBP Increases Solubility of Proteins To Which It Is Fused E. coli maltose-binding protein (MBP) has been observed to increase the solubility of its fusion partners (Kapust & Waugh 1999) MBP may act as a general molecular chaperone (Kapust & Waugh 1999) MBP possesses protein binding sites Mutational analyses reveal critical residues for native protein stability and solubilizing activity

8. MBP Tac promoter (A) MBP target TEV cleavage site (B) 6X-His tag attR recombinational cloning sites Gateway-MBP System Gateway-MBP is a ligation-independent cloning system for expression of MBP fusions in E. coli

9. Cleavage by TEV MBP is cleaved from its fusion partner by Tobacco Etch Virus (TEV) Protease TEV recognizes the consensus sequence: Glu-X-X-Tyr-X-Gln-Ser (Routzahn & Waugh 2002) Both in vivo and in vitro cleavage conditions are being investigated Interesting note: Recombinant TEV does not fold properly in vivo and it must be generated as an MBP fusion as well (Dougherty et al., 1989)

10. MBP-fusion Expression & Solubility Screening 24 C. elegans targets Compared expression and solubility levels among: pET14 pET15 MBP in vivo cleavage MBP in vitro cleavage Analysis by SDS-PAGE

11. BL21 pRK603 pKC1BL21 (DE3) pMgk DE3 pMgK pMBP pKC1 pRK603 pRK603: MBP-TEV fusion pKC1: rare codon tRNAs pMgk: rare codon tRNAs In vivo cleavage of MBP In vitro cleavage of MBP Two E. coli strains must be used

12. Expression & Solubility Analysis of MBP-fusion Proteins WR3 is insoluble when expressed in pET14/15 constructs MBP-WR3 is highly soluble when cleaved in vivo and in vitro in vivo in vitro Total (T) Soluble (S)T MBP- WR3 MBP S S P/C WR3 T MBP

13. Summary of MBP-fusion Screening

14. From pET to MBP: the fates of our targets pET14/15 Expression & Solubility Analysis

15. From pET to MBP: with in vivo cleavage Not expressed as pETExpressed/not soluble as pET Expressed/soluble as pET

16. 1/3 cleaved & soluble 2/3 low concentration 1/6 cleaved & soluble 2/6 not purified 3/6 low concentration 6/8 cleaved & soluble 2/8 low concentration Not expressed as pET Expressed/soluble as pET (3) (5) 100% (8) (1) (6) Expressed/not soluble as pET From pET to MBP: with in vitro cleavage

17. SUMO Small ubiquitin-related modifier, 11 kDa, highly soluble, globular protein SUMO has varied biological functions including cellular localization and transcriptional regulation Fusion to ubiquitin has been shown to increase recombinant protein solubility in both E. coli and S. cerevisiae (Butt et al., 1989; Ecker et al., 1989) ubiquitin Ulp1-Smt3 complex (Mossessova & Lima, 2000)

18. SUMO MCS T7 promoter BsaI/BsmBI XhoI/HindIII SUMO System Utilizes class IIS restriction endonucleases BsaI recognition sequence: 5’ GGTCTCNNNNNN 3’ CCAGAGNNNNNN 3x4ul 3hr 2x5ul 4hr Cleavage can be improved, but is not complete

19. SUMOtarget Ulp1 cleavage site 6X-His tag Ulp1 separates protein target from the fusion by recognizing the entire SUMO protein SUMO System Cleavage with Ulp1 will leave no extra residues at the N-terminus of the target

20. SUMO system utilizes Ni-affinity chromatography Cleavage must occur in vitro Development of robotic methods necessary for purification steps SUMO system (Figure courtesy of www.lifesensors.com)

21. SUMO-fusion Expression & Solubility Screening 50 targets among: A. thaliana D. melanogaster C. elegans H. sapiens 8 positive clones screened Compared expression and solubility levels among: pET14/15/21 SUMO-fusions uncleaved Analysis by SDS-PAGE

22. 6 14 18 28 38 49 62 Gel 1Gel 2 2 3 4 5 6 7 8 9 10 11 2 3 4 5 6 7 MW Expression & Solubility Analysis of SUMO-fusion Proteins

23. Summary of SUMO-fusion Screening 50% increase in expressed and soluble proteins when fused with SUMO

24. Conclusions Protein insolubility and inclusion body formation is a major barrier to high-throughput structural genomics efforts New methods, which allow for labeling of proteins,must be developed for expression of soluble eukaryotic proteins Thus far, MBP appears to have significant solubilizing activity. Partner proteins are soluble after cleavage, suggesting some type of chaperone effect Uncleaved SUMO-fusions exhibit high expression and increased solubility levels

25. Future Prospects Complete cloning of targets (currently underway) 8/50 successfully cloned Complete expression & solubility screens including cleavage with Ulp1 Optimize robotic 96-well purification protocols MBP Complete screening for an additional 26 targets Optimize in vitro cleavage with TEV SUMO After screening is complete, comparisons can be made about the types of proteins that are rescued. We can then use bioinformatic techniques to identify other potential targets.

26. Butt, TR, Jonnalagadda, S, Monia, BP, Sternberg, EJ, Marsh, JA, Stadel, JM, Ecker, DJ, and Crooke, ST. (1989) Proc. Natl. Acad. Sci. 86, 2540-2544. Dougherty, WG, Cary, SM, and Parks, TD. (1989) Virology 171, 356-364. Ecker, DJ, Stadel, JM, Butt, TR, Marsh, JA, Monia, BP, Powers, DA, Gorman, JA, Clark, PE, Warren, F, Shatzman, A, and Crooke, ST. (1989) J. Biol. Chem. 264, 7715-7719. Edwards, AM, Arrowsmith, CH, Christendat, D, Dharamsi, A, Friesen, JD, Greenblatt, JF, and Vedadi, M. (2000) Nature Struct. Biol. 7 (suppl), 970-972. Kapust, RB and Waugh, DS. (1999) Prot. Sci. 8, 1668-1674. Mossessova, E and Lima, CD. (2000) Mol. Cell 5, 865-876. Routzahn, KM and Waugh, DS. (2002) Journal of Structural and Functional Genomics 2, 83-92. References

27. Acknowledgements Gaetano Montelione Tom Acton Ritu Shastry Chi Kent Ho Li-Chung Ma Yiwen Chiang Rong Xiao Thank you for all your encouragement, assistance, and support!