1. A comparison of multiple E. coli protein expression systems using orthologous plant proteins Belinda Sharpe Division of Biology Faculty of Natural Sciences Imperial College London

2. EDS1 Functions as part of a signalling pathway in plant defence responses Part of a family of related proteins, including PAD4 and SAG101 All share a similar domain structure from sequence homology EDS1 can physically interact with PAD4, SAG101, and also with itself lipase-like domainEP domain

3. EDS1 Initially focussed on full-length EDS1, aiming towards crystallisation trials Full length AtEDS1 with N-terminal His-tag (pET15b) High levels of expression (~30 mg/L), expressed in both soluble and insoluble fractions Purification of soluble protein was successful, and appeared monomeric by gel filtration Was not stable over time and no success in crystallisation trials EDS1

4. EDS1-SAG101 complex Given EDS1 was unstable in isolation, attempt co-expression of EDS1 with protein binding partners (PAD4 and SAG101) Co-expression and co-purification of EDS1 with His-tagged SAG101 was successful However, the complex precipitated in crystallisation trials EDS1 SAG101 97 66 45 kDa

5. Fragments of EDS1 ~40 kDa fragment ~30 kDa fragment 3541 80 623 Full-length EDS1 (73 kDa) ~40 kDa fragment ~30 kDa fragment Use limited proteolysis to identify smaller stable fragments of EDS1 Two stable fragments were identified using combination of N-terminal sequencing, MS, and size estimation by SDS-PAGE 354

6. Next step… 1.Compare expression and solubility of full length EDS1 and smaller defined EDS1 fragments 2.Use different expression vectors, with different affinity tags 3.Compare Arabidopsis EDS1 to another EDS1 orthologue

7. 1. EDS1 constructs ~40 kDa ~30 kDa 354 1 80 623 1 354 ~73 kDa EDS1- Full EDS1- Long EDS1- Short

8. 2. New expression vectors Gateway system Based on in vitro site- specific recombination system Allows you to move your gene of interest into one “entry” vector, then easily into multiple vector systems for functional analysis and protein expression

9. 2. New expression vectors GSTEDS1 construct A TEV protease site was engineered into the sequence, to allow for cleavage of each tag if required EDS1 construct 6xHis MBP EDS1 construct 6xHis

10. 3. Choosing an orthologue EDS1 is found in Arabidopsis, tomato, tobacco, barley, wheat, rice, and others. EDS1 in tomato has been shown to have the same function as EDS1 in Arabidopsis. Sequences are 60% similar, 42% identical.

11. Summary of constructs Arabidopsis partly soluble Tomato 6xHisGST MBP 6xHis EDS1S EDS1L EDS1F EDS1S EDS1L EDS1F

12. Induction conditions Various induction conditions trialled  IPTG concentration (50 μM – 1 mM) Made no significant difference to levels of expression  Temperature (20 and 37°C) Constructs were completely insoluble at 37°C, however some soluble protein was observed at 20°C

13. Comparison of EDS1 constructs Although His-tagged full-length EDS1 was partly soluble, His-tagged smaller domains of EDS1 were not. EDS1SEDS1LEDS1F solins 6xHis 97 66 45 30 kDa insoluble partly soluble

14. Comparison of vectors 6xHisGST MBP 6xHis Soluble protein for HisMBP construct only 97 66 45 30 kDa insoluble partly soluble EDS1L

15. Comparison of orthologues Differences between soluble protein expression for Arabidopsis and tomato constructs were seen for the HisMBP-EDS1L fusion protein MBP EDS1L 6xHis insoluble partly soluble

16. Summary of results Arabidopsis insoluble partly soluble insoluble partly soluble Tomato insoluble partly soluble 6xHisGST MBP 6xHis EDS1S EDS1L EDS1F EDS1S EDS1L EDS1F

17. Summary Did see difference in expression of soluble protein between different constructs, vectors, and between Arabidopsis and tomato Given initial results for full-length EDS1, there was no dramatic shift in the percentage of soluble and insoluble protein expressed Next… look at other tags (pET32-Trx), and explore expression in a different system to E.coli

18. Thanks to… Bart Feys Nick Brereton Vincenzo Salerno