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R Pharmaceuticals D. Ambrosius; slide 1 Proteine/RAMC-Presentation-9-01 Various Strategies Used to Obtain Proteins for Crystallization and Biostructural.

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Presentation on theme: "R Pharmaceuticals D. Ambrosius; slide 1 Proteine/RAMC-Presentation-9-01 Various Strategies Used to Obtain Proteins for Crystallization and Biostructural."— Presentation transcript:

1 r Pharmaceuticals D. Ambrosius; slide 1 Proteine/RAMC-Presentation-9-01 Various Strategies Used to Obtain Proteins for Crystallization and Biostructural Studies Dorothee Ambrosius, R. Engh, F. Hesse, M. Lanzendörfer, S. Palme, P. Rüger Roche Pharmaceutical Research, Penzberg

2 r Pharmaceuticals D. Ambrosius; slide 2 Proteine/RAMC-Presentation-9-01 Protein Classes extracellular proteins  plasma protein concentration:  70 mg/ml transporter (albumin) immuno-globulin enzymes, enzyme-inhibitors coagulation factors, lipoproteins  protein characteristics/ stability often monomeric proteins contain disulfide bridges protease resistant stable fold intracellular proteins  cytoplasma and organelles:  300-800 mg/ml multi-enzyme complexes enzyme cascades transcription complexes focal adhesion/integrins cytoskeleton, heat-shock proteins  protein characteristics/stability often multimeric complexes no disulfide bridges very labile proteins ; short half-life require stabilization: interaction with other proteins

3 r Pharmaceuticals D. Ambrosius; slide 3 Proteine/RAMC-Presentation-9-01 Protein Sources/Expression Systems

4 r Pharmaceuticals D. Ambrosius; slide 4 Proteine/RAMC-Presentation-9-01 Biological Function of Cytokines G-CSF Neutrophils Source: Herrmann/Lederle

5 r Pharmaceuticals D. Ambrosius; slide 5 Proteine/RAMC-Presentation-9-01 Development Goals for Recombinant Human G-CSF  native sequence:  without additional N- terminal Met  reduction of immunogenicity risk  potency:  equal to Amgen´s Neupogen  low production cost:  E. coli as host strain  in vitro refolding  consistent quality:  robust downstream scheme  analytical methods established  Hu-G-CSF:hematopoietic growth factor (174 aa) 2 S-S bridges, one single Cys 17  Clinical use: patients with neutropenia: after chemotherapy  improved haemotopoietic recovery  reduction of infectious risks

6 r Pharmaceuticals D. Ambrosius; slide 6 Proteine/RAMC-Presentation-9-01 Genetic engineering of an economic downstream process Strategy: Development of Recombinant Human G-CSF Fusion Peptide  high level expression  improved refolding  efficient separation of cleaved and uncleaved protein  optimized cleavage site Human G-CSFFusion Peptide Protease  specific  efficient  recombinant  consistent quality rhG-CSF  low production costs  without N-terminal Met  equal potency/efficiency  consistent quality  improved quality 

7 r Pharmaceuticals D. Ambrosius; slide 7 Proteine/RAMC-Presentation-9-01 Cleavage - ++ + +++ ++ +++ Expression (%) 100 30 100 25 10 100 Renaturation (%) 10 20 50 90 80 Fusion Peptide Met G-CSF Met-Thr-Pro-Leu  G-CSF Met-Thr-Pro-Leu-His-His  G-CSF Met-Thr-Pro-Leu-Lys-Lys  G-CSF Met-Thr-Pro-Leu-Glu-Glu-Gly  G-CSF Met-Thr-Pro-Leu-Glu-Glu-Gly-Thr-Pro-Leu  G-CSF Met-Lys-Ala-Lys-Arg-Phe-Lys-Lys-His  G-CSF  Cleavage Site (Pro-Arg-Pro-Pro) Optimization of rhG-CSF Fusion Proteins Source: EP 92102864.3 ; DE 4104580

8 r Pharmaceuticals D. Ambrosius; slide 8 Proteine/RAMC-Presentation-9-01 Refolding Kinetics of rhG-CSF Fusion Protein Solubilization 6,0 M Gdn/HCl, pH 8.0 100 mM Tris,/HCl 100 mM DTE 1 mM EDTA Temperature: RT c= 20 mg/ml Renaturation 0,8 M Arginine/HCl 100 mM Tris/HCl, pH 8.0 0.5 / 0.5 mM = GSH / GSSG 10 mM EDTA Temperature: RT Protein conc. 0.5 -1.0 mg /ml Time: 1- 2 hours native denat. Source: EP 92102864.3 ; DE 4104580 Pellet SN

9 r Pharmaceuticals D. Ambrosius; slide 9 Proteine/RAMC-Presentation-9-01 Role of p53 in cell cycle control:“guardian of the genome” latent p53 active p53 activation accumulation h stress factors or oncogenic proteins mdm2 cell type level of p53 extent of DNA damage genetic background cell cycle arrest: repair defective genes apoptosis: kill harmful deregulated cells negative feedback loop !!

10 r Pharmaceuticals D. Ambrosius; slide 10 Proteine/RAMC-Presentation-9-01 Engineering of MDM2 for biostructural purposes The MDM2 oncoprotein is a cellular inhibitor of the p53 tumor suppressor. Goal:Improvement of biophysical properties of HDM2 (human MDM2) by “crystal engineering” Known:  XDM2 (Xenopus laevis MDM2): - better solubility, suitable for biostructural investigations - wrong species and reduced binding affinity  HDM2 (25-108): - high binding affinity to p53 peptide - prone to aggregation, not suitable for biostructural studies Strategy:  use XDM2 as scaffold and humanize its p53-binding site  introduce point mutations in HDM2 to increase solubility  remove flexible ends at both sides of structured p53- binding region

11 r Pharmaceuticals D. Ambrosius; slide 11 Proteine/RAMC-Presentation-9-01 Figures taken from Kussie et al., Science 274 (1996) 948. Structure of MDM2/p53-peptide complex Resolution X-ray structures: human MDM2/p53: 2.6 Å Xenopus MDM2/p53: 2.3 Å p53 mdm2 17-29 26-108

12 r Pharmaceuticals D. Ambrosius; slide 12 Proteine/RAMC-Presentation-9-01 MDM2 variants created by protein engineering human MDM2 1 26 108 125 185 240 300 330 350 440 491 p53 binding HDM2 (17-125)X-ray published HDM2 (25-108)X-ray HDM2 (25-108) mutants X-ray XDM2 (13-119)X-ray published, NMR XDM2 (13-119) LHINMR, X-ray XDM2 (21-105) LHIX-ray I50L P92H L95I

13 r Pharmaceuticals D. Ambrosius; slide 13 Proteine/RAMC-Presentation-9-01 Step 15 N-labeled non-labeled (LB) (minimal medium) Fermentation10 L 10 L E. coli (wet weight)90 g 600 g Inclusion bodies (w.w.) 3.5 g 85 g IB total protein content 1.3 g 30 g MDM2 (50-70% yield) 0.8 g 18 g Renaturation (~25%) 0.2 g 4.5 g MDM2 (Purification) 0.16 g 3.6 g Final product 0.1 g 2.2 g Human MDM2: Yields & Upscale

14 r Pharmaceuticals D. Ambrosius; slide 14 Proteine/RAMC-Presentation-9-01 Crystals of hXDM/peptide Some crystals comply with corporate identity rules hXDM2/p53 peptide Patience might be rewarded Conditions: 0.1 M MES pH 6.2, 4.0 M NaOOCH 3 days after micro seeding at 13 °C 4 months at 4 °C hXDM2/phage-peptide

15 r Pharmaceuticals D. Ambrosius; slide 15 Proteine/RAMC-Presentation-9-01 I: Ser/Thr-Kinase FamiliesSubfamilies/Structures Ia: Non Receptor Ser/Thr-Kinase familiy cAPK: cAMP dependent protein kinase PKA, PKB, PKC cdks: Cyclin dependent kinase cdk2, cdk4, cdk6 MAPK: Mitogen activated protein kinase Erk, Erk2, Jnk, p38( , ,  ) MLCK: Myosine light chain kinase Twitchin, Titin CK: Casein kinase Ck-1, Ck-2 PhK: Phosphorylase kinase (tetramer: , , ,  ) PhK CaMK: Calcium/calmodulin dependen kinase CaMK Ib: Receptor Ser/Thr-Kinase family TGF1-R KinaseTGF1-ßR II: Tyr-Kinase FamiliesSubfamilies/Structures IIa: Non receptor Tyr-Kinase family SRC-family SRC, c-SRC, CSK, HCK LCK: humam lymphocyte kinase: LCK, c-Abl IIb: Receptor Tyr-Kinase family EGFR-family:EGFR, ErbB2-4 InsR-familyIRK, IGF1R, IRR PDGFR-, CSFR-, Met-, Ron-familiy, FGF1-R, VEGFR-K EphA1….EphB1, Trk A, B, C, etc. Protein Kinase Families (incomplete list)

16 r Pharmaceuticals D. Ambrosius; slide 16 Proteine/RAMC-Presentation-9-01 PKA: 2 Å X-ray Structure Further details for crystallization see poster of Ch. Breitenlechner

17 r Pharmaceuticals D. Ambrosius; slide 17 Proteine/RAMC-Presentation-9-01 PKA: cyclic AMP Dependent Protein Kinase Expression:  E. coli, solubly expressed in phosphorylated, active form  20-50 mg purified protein (10 l fermentation) Purification:  affinity chromatography with inhibitory peptide (PKI) mimicking substrate binding  Ref.: R. Engh & D. Bossemeyer, Adv. Enz. Reg. 41, 2001 Binding Affinity:  20 nM of inhibitory peptide (PKI) Protein:  MW: 35 kDa  Ser/The kinase  monomeric 2 domain (C- and N-lobe) protein without additional regulatory domains (SH2, SH3, etc.)  extended structured C- and N-Terminus, which possibly stabilizes the overall kinase structure Ideal model: Ser/Thr protein kinase inhibitor studies generation of other Ser/The kinase (e.g. PKB, Aurora) structures

18 r Pharmaceuticals D. Ambrosius; slide 18 Proteine/RAMC-Presentation-9-01 Major Components of the Cell Cycle Machinery  mitogen induced progression through the cell cycle requires timely controlled activation of different cyclin-dependent kinases (CDKs)  cyclins (D, E, A, B), periodically expressed throughout the cycle, are the regulatory subunits of CDKs (activation)  members of the p16(INK4)- and p21(KIP)-protein family inhibit CDKs and CDK-cyclin complexes and arrest inappropriate cell cycle progression G1G1 S M G2G2 Cell Cycle G0G0 CDK2 cyclin A CDC2 cyc. A/B CDK2 cyclin E CDK4/6 cyclin D CDC2 cyclin B Mitosis DNA Replication INK4 Kip/ Cip Kip/ Cip

19 r Pharmaceuticals D. Ambrosius; slide 19 Proteine/RAMC-Presentation-9-01 Cyclin Dependent Kinases: CDK2 and CDK4/6 N. Pavletich, JMB 287, 821-828, 1999

20 r Pharmaceuticals D. Ambrosius; slide 20 Proteine/RAMC-Presentation-9-01 Structural investigations of cdks (incomplete list)

21 r Pharmaceuticals D. Ambrosius; slide 21 Proteine/RAMC-Presentation-9-01 Summary  Proteins show a tremendous diversity with respect to - biological function and cellular location - structure, conformation and stability  E. coli is a very attractive expression system with respect to time, yield, costs and production of isotope labeled proteins  Application of in vitro protein refolding is a powerful tool to generate native structured proteins and should be considered as alternative  The protein kinase family is regulated by multiple mechanism and show conformational diversity of catalytic cores; high degree of flexibility - e.g. IRK(3P) and LCK (Tyr kinases) show structural homology to cAPK and cdks (Ser/Thr kinases)  Until today, most kinases successfully applied for structural research are expressed as active P--enzyme in baculo/insect cells; exception PKA

22 r Pharmaceuticals D. Ambrosius; slide 22 Proteine/RAMC-Presentation-9-01 Acknowledgement PEX:S. Kanzler, H. Brandstetter (MPI) MDM2:G. Saalfrank, Ch. Breitenlechner (MPI), U. Jacob (MPI) IL-16: B. Essig, P. Mühlhahn (MPI), T. Holak (MPI) MIA: G. Saalfrank, C. Hergersberg, R. Stoll (MPI), T. Holak (MPI) cAPK:G. Achhammer, E. Liebig, Ch. Breitenlechner (MPI) cdks: H. Hertenberger, J. Kluge, U. Jucknischke G-CSF: S. Stammler, M. Leidenberger, U. Michaelis, T. Zink (MPI), T. Holak (MPI)

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