Rapid production of fusion proteins using the RTS 100/500 Systems

1. 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 J. Stracke, 09/01

2. 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) J. Stracke, 09/01

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

4. 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 Classical Protein Production J. Stracke, 09/01

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

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

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

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

9. Successfully crystallised proteins Crystals of phosphoserine phosphatase (PSP) 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 His6 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. J. Stracke, 09/01

10. RTS 100 expression • in vitro batch expression in 50µlreactions • 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) J. Stracke, 09/01

11. promotor module terminator module cs tag T7T T7P RBS tag cs gene X PCR gene X cs tag T7T T7P RBS tag cs 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! RTS 100 expression Schematic illustration of template generation for RTS 100 expression using PCR J. Stracke, 09/01

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

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

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

15. 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 PEX (MMP2) 4 1 TIMP2 4 1 N 3 2 C 2 3 Gohlke et al. (1996) Williamson et al. (1994) Example 1 - Structures of MMP2-PEX and TIMP2 J. Stracke, 09/01

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

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

18. 80 Dissociation Association 70 60 50 40 TIMP-2 concentration Response Difference [RU] 30 20 10 Ligand : PEX2_ NtAT 0 Analyte : TIMP2 Injection -10 300 100 200 250 350 400 -50 0 50 150 Time [s] Equilibrium constant KD = 1.5x 10-10M Example 1 - Evaluation of constructs for BIAcore analysis Analysis of ligand interactions of RTS-expressed PEX2-NtAT (N-terminaler AVITAG) with TIMP-2 using BIAcore J. Stracke, 09/01

19. Example 2 - Production of active Protein B Aim: Production of sufficient quantities of active Protein B for biochemical characterisation and crystallisation. • 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. J. Stracke, 09/01

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

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

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

23. Conclusions • 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 Overcoming the bottleneck of protein production for crystallography seems possible in the near future using RTS! J. Stracke, 09/01

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