Protein Expression and Folding Optimization For High-Throughput Proteomics

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

2. Introduction • 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. • 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) • New expression technologies are necessary • to provide high quality, labeled, recombinant • eukaryotic proteins suitable for structural studies.

3. Protein Production Pipeline PCR & cloning of ORFs Construct validation Small-scale expression & solubility screens Large-scale production & purification of labeled proteins 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 Attempt to use fusion protein expression systems in place of our standard T7 Multiplex Expression Systemfor a set of 50 eukaryotic target proteins 1) Gateway MBP-fusion expression system (Kapust & Waugh 1999) 2) SUMO system (Lifesensors, Inc. Malvern, PA)

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

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 (Dougherty et al., 1989) Both in vivo and in vitro cleavage conditions are being investigated (Routzahn & Waugh 2002) Interesting note: Recombinant TEV does not fold properly in vivo and it must be generated as an MBP fusion as well

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

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

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 pET Expressed/not soluble as pET Expressed/soluble as pET

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

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

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

20. SUMO system • SUMO system utilizes • Ni-affinity chromatography • Cleavage must occur in vitro • Development of robotic methods • necessary for purification steps (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. Expression & Solubility Analysis of SUMO-fusion Proteins 2 3 4 5 6 7 2 3 4 5 6 7 8 9 10 11 62 49 38 MW 28 18 14 6 Gel 1 Gel 2

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 MBP • Complete screening for an additional 26 targets • Optimize in vitro cleavage with TEV SUMO • Complete cloning of targets (currently underway) • 8/50 successfully cloned • Complete expression & solubility screens • including cleavage with Ulp1 • Optimize robotic 96-well purification protocols 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. References 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) Virology171, 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. Cell5, 865-876. Routzahn, KM and Waugh, DS. (2002) Journal of Structural and Functional Genomics2, 83-92.

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!