1. r Pharmaceuticals J. Stracke, 09/01 Rapid production of fusion proteins using the RTS 100/500 Systems Jan Stracke, Michael Schräml, Andreas Junger, Dorothee Ambrosius and Martin Lanzendörfer Roche Diagnostics GmbH, Pharmaceutical Research, Dept. of Biochemistry, Penzberg, Germany

2. r Pharmaceuticals J. Stracke, 09/01 Protein Production High need for purified proteins in pharmaceutical research for: X-ray crystallography (X-ray) Nuclear Magnetic Resonance (NMR) High Throughput Screening (HTS) Therapeutic Proteins (e.g. antibodies)

3. r Pharmaceuticals J. Stracke, 09/01 Bottlenecks of protein crystallisation Gene Protein Crystal/ Structure Screening of various constructs for expression Screening of various muteins and/or truncated constructs for crystallisation

4. r Pharmaceuticals J. Stracke, 09/01 Classical Protein Production cloning expression fermentation cell lysis soluble / insoluble protein refolding purification characterisation activity assays, interaction analysis high need for acceleration of protein production high need for acceleration of methods time consuming step

5. r Pharmaceuticals J. Stracke, 09/01 Rapid Translation System (RTS) gene DNA-template T7-RNA polymeraseE.coli lysate protein mRNA gene RTS 100RTS 500RTS 500 HY 50 µg / ml500 µg / ml5 mg / ml control of expression by SDS-PAGE, Western blotting, ELISA transcription & translation RTS 500 RTS 100

6. r Pharmaceuticals J. Stracke, 09/01 Proteins successfully expressed with RTS Endostatin Erythropoietin Interleukin-2 p40phox SH3 mod. Phosphodiesterase Rab 5 VEGF-receptor Green fluorescent p. b -Galactosidase HIV Tat s-Adenosylmethio- nine synthetase 1 Protein MW 20 30 15.4 47 56 22 41 28 118 10 41.6 Organism human animal bacteria virus yeast Function Inhibitor hormone regulatory protein GTPase receptor fragment fluorescent protein enzyme transcription factor enzyme Yield RTS 500 [µg] 100 550 600 90 100 250 560 105 n. d. 6

7. r Pharmaceuticals J. Stracke, 09/01 Successfully expressed proteins - a system comparison RTS 500 vs RTS 500 HY

8. r Pharmaceuticals J. Stracke, 09/01 Further applications of RTS Expression of......cell toxic proteins…...two proteins in parallel... …in the RTS 500.

9. r Pharmaceuticals J. Stracke, 09/01 The protein was expressed using the RTS 500 E. coli HY Kit. Approximately 3 mg of PSP protein, fused at the C-terminus with a His 6 tag, was produced in a single 1 ml reaction, purified, concentrated and subsequently crystallised at 25°C using the hanging-drop method. Data courtesy of Ho S. Cho, Weiru Wang, Sung-Hou Kim and David E. Wemmer, Berkeley Structural Genomic Center. Crystals of phosphoserine phosphatase (PSP) Successfully crystallised proteins

10. r Pharmaceuticals J. Stracke, 09/01 RTS 100 expression in vitro batch expression in 50 µl reactions designed for the use of linear PCR-generated DNA templates enables the fast and parallel expression and evaluation of numerous constructs (e.g. for crystal engeneering)

11. r Pharmaceuticals J. Stracke, 09/01 gene X T7P RBS tag cs PCR cs tag T7T gene X cs tag T7T T7P RBS tag cs promotor module terminator module linear template for in vitro translation (batch) Various promotor and terminator modules can be prepared and fused by PCR in numerous combinations with the gene of interest. Expression of these constructs is performed in the cell-free translation system RTS 100. MTP format is possible! Schematic illustration of template generation for RTS 100 expression using PCR RTS 100 expression

12. r Pharmaceuticals J. Stracke, 09/01 Successfully expressed proteins - a system comparison RTS 100 HY vs RTS 500 HY lower yields in the RTS 100 compared to RTS 500

13. r Pharmaceuticals J. Stracke, 09/01 High Throughput Protein Production (HTPP) using RTS 100 goal acceleration of protein production PCR product (e.g. RT-PCR) RTS (in vitro expression) soluble / insoluble protein refolding high affinity purification activity assays, interaction analysis production in days (not months) high throughput high flexibility automation small scale production need for high yield expression classical or in vitro optimisation MTP-96 well-format

14. r Pharmaceuticals J. Stracke, 09/01 Evaluation of: Solubility Affinity-tags (purification, immobilisation) Ligand activity (BIAcore) using the RTS 500 system for construct expression. Example 1

15. r Pharmaceuticals J. Stracke, 09/01 Example 1 - Structures of MMP2-PEX and TIMP2 MMP2-PEX: hemopexin-like domain of MMP2 MW = 23 kDa; 1 disulfide bridge TIMP2: tissue type inhibitor of matrixmetallo-proteinase 2 MW = 22 kDa; 6 disulfide bridges Gohlke et al. (1996) PEX (MMP2) 1 2 3 4 1 23 4 N C TIMP2 Williamson et al. (1994)

16. r Pharmaceuticals J. Stracke, 09/01 4 protein coding genes in 7 constructs promotorfused genes tac PinPoint-tag PEX2 PinPoint-tag TIMP2 tac PinPoint-tag Protein A tac AVITAG PEX2 T7 Protein B AVITAG T7 Strep-tag PEX2 T7 Poly-Glu - -tag PEX2 T7 E.coli RTS-500 and E.coli vector Xa3-PinPoint pIVEX 2.1 pIVEX 2.2b Nde pIVEX 2.3 MCS Example 1 - Evaluation of constructs for BIAcore analysis

17. r Pharmaceuticals J. Stracke, 09/01 Relative yields of expressed protein - Improvement of solubility in the RTS-500 System E.coliRTS-500 System 0 % Protein A NtPP Protein B NtAT Example 1 - Evaluation of constructs for BIAcore analysis

18. r Pharmaceuticals J. Stracke, 09/01 Analysis of ligand interactions of RTS-expressed PEX2-NtAT (N-terminaler AVITAG) with TIMP-2 using BIAcore Example 1 - Evaluation of constructs for BIAcore analysis

19. r Pharmaceuticals J. Stracke, 09/01 Example 2 - Production of active Protein B Evidence exists that: a truncated form may be active or co-factors may be required for activity These aspects are being addressed using the RTS 100 and 500 systems for construct expression/evaluation. Aim: Production of sufficient quantities of active Protein B for biochemical characterisation and crystallisation.

20. r Pharmaceuticals J. Stracke, 09/01 Example 2 - Production of active Protein B truncated/mutated constructs by PCR activity assay expression in 96 well plates (RTS 100) target protein expression (RTS 500) immobilisation ligand fishing co-expression with selected activating proteins (e.g. RTS 100) activity assay RT-PCR ligand identification Active Protein B Truncations/mutations Co-expression

21. r Pharmaceuticals J. Stracke, 09/01 Example 2 - Production of active Protein B Truncated Protein B constructs, generated by PCR for expression in the RTS 100 Prot. B gene B cs tag T7T T7P RBS tag cs

22. r Pharmaceuticals J. Stracke, 09/01 Example 3 - Production of various muteins Aim: Evaluation of approx. 50 muteins of Protein C (extracellular, 7 disulphide bonds) for crystallisation. Strategy: generation of numerous mutated linear constructs by PCR assay for ligand activity (e.g. BIAcore) selection of active and inactive constructs for large scale expression and crystallisation expression in 96 well plates (RTS 100) refolding and purification in 96 well plates info about ligand binding site

23. r Pharmaceuticals J. Stracke, 09/01 proteins of interest can be expressed in an active, soluble form in the RTS systems in µg/mg quantities for initial characterisation (e.g. activity) proteins can easily be co-expressed if necessary due to the possibility to use linear PCR products as templates, the RTS 100 allows rapid parallel screening of numerous protein constructs with regard to activity, ligand binding and solubility RTS 100 is predestinated for automation of protein expression and purification Conclusions Overcoming the bottleneck of protein production for crystallography seems possible in the near future using RTS!

24. r Pharmaceuticals J. Stracke, 09/01 Acknowledgements Roche Pharma Research A. Grossmann R. Engh M. Dangl P. Rüger K. Lang Roche Mol. Biochemicals C. Nemetz A. Gräntzdörfer S. Wessner M. Watzle Max Planck Inst.f. Biochemie R. Engh R. Huber N. Heim M. Wisniewska TU-München J. Buchner