r pharmaceuticals d. ambrosius; slide 1 proteine/ramc-presentation-9-01 various strategies used to...

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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

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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

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Protein Sources/Expression Systems

Expression system Advantages Examples Structure

E. coli soluble inclusion bodies

rapid cloning/ expression high yield isotope labeling possible

G-CSF; IBsPEX, IBsMIA, IBsIL-16, solubleMDM2, IBsPKA, soluble

NMRX-rayNMRNMRX-ray, NMRX-ray

Baculo/Insect cells

expression of active protein modifications

most Tyr kinases(RTK: IRK,c-met,SRC, LCK, etc.)Ser/Tyr kinasese.g. cdks, cAPK

X-ray/NMR

X-ray/NMR

RTS: E. coli parallel expression high throughput proteomics

see talk & posterJ . Stracke

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Biological Function of Cytokines

G-CSFNeutrophils

Source: Herrmann/Lederle

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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

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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

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Cleavage

-

++

++

+

+++

++

+++

Expression

(%)

100

30

100

100

25

10

100

Renaturation

(%)

10

20

20

50

90

80

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

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Refolding Kinetics of rhG-CSF Fusion Protein

Solubilization6,0 M Gdn/HCl, pH 8.0 100 mM Tris,/HCl100 mM DTE 1 mM EDTATemperature: RTc= 20 mg/ml

Renaturation0,8 M Arginine/HCl100 mM Tris/HCl, pH 8.00.5 / 0.5 mM = GSH / GSSG10 mM EDTATemperature: RTProtein conc. 0.5 -1.0 mg /mlTime: 1- 2 hours

native

denat.

Source: EP 92102864.3 ; DE 4104580

Pellet SN

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Role of p53 in cell cycle control:“guardian of the genome”

latent p53 active p53

activationaccumulation

h

stress factorsor 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 !!

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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

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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

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MDM2 variants created by protein engineering

human MDM21 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) LHI NMR, X-ray

XDM2 (21-105) LHI X-ray

I50L P92H

L95I

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Step 15N-labeled non-labeled (LB)

(minimal medium)

Fermentation 10 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

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Crystals of hXDM/peptide

Some crystals comply withcorporate 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

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I: Ser/Thr-Kinase Families Subfamilies/StructuresIa: Non Receptor Ser/Thr-Kinase familiy

cAPK: cAMP dependent protein kinase PKA, PKB, PKCcdks: Cyclin dependent kinase cdk2, cdk4, cdk6

MAPK: Mitogen activated protein kinase Erk, Erk2, Jnk, p38(,,)

MLCK: Myosine light chain kinase Twitchin, TitinCK: Casein kinase Ck-1, Ck-2PhK: Phosphorylase kinase (tetramer: , , , ) PhK

CaMK: Calcium/calmodulin dependen kinase CaMK

Ib: Receptor Ser/Thr-Kinase familyTGF1-R Kinase TGF1-ßRII: Tyr-Kinase Families

Subfamilies/StructuresIIa: Non receptor Tyr-Kinase family

SRC-family SRC, c-SRC, CSK, HCK

LCK: humam lymphocyte kinase: LCK, c-Abl

IIb: Receptor Tyr-Kinase familyEGFR-family: EGFR, ErbB2-4InsR-family IRK, IGF1R, IRRPDGFR-, CSFR-, Met-, Ron-familiy, FGF1-R, VEGFR-KEphA1….EphB1, Trk A, B, C, etc.

Protein Kinase Families (incomplete list)

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PKA: 2 Å X-ray StructureFurther details for crystallization see poster of Ch. Breitenlechner

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PKA: cyclic AMP Dependent Protein KinaseExpression: 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

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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

G1

S

M

G2

Cell Cycle

G0

CDK2

cyclin A

CDC2

cyc. A/B CDK2

cyclin E

CDK4/6

cyclin D

CDC2

cyclin BMitosis

DNA Replication

INK4

Kip/Cip

Kip/Cip

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Cyclin Dependent Kinases: CDK2 and CDK4/6

N. Pavletich, JMB 287, 821-828, 1999

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Structural investigations of cdks (incomplete list) Structure Method Protein Expression system Referencep16p16

Folding studiesNMR

p16GST-p16

E. coli (IBs)E. coli (soluble)

Tang, 1999Byeon, 1998

p18

p18

NMR

X-ray: 1.95 Å

GST-p18

p18

E. coli (soluble)

BL21 (soluble)

Yuan, 1999

Venkataramani, 1998p19 NMR p19 E. coli (IBs) Baumgartner, 1999p19/cdk6, p16/cdk6

p19/cdk6

X-ray: 2.8 ÅX-ray: 3.4 Å

X-ray: 1.9 Å

cdk6GST-p19/p16

p19GST-cdk6

Baculo/insect cellsE. coli (soluble)

E. coli (soluble)Baculo/insect cells

Russo, 1998

Brotherton, 1998p18/cdk6/cycK X-ray: 2.9 Å GST-cycK

GST-p18cdk6

E. coli (soluble)E. coli (soluble)Baculo/insect cells

Jeffrey, 2000

cycA-cdk2cycA-ATPS-cdk2

X-ray: 2.3 ÅX-ray: 2.6 Å

cdk2cycA:

Baculo/insect cellsE.coli (soluble)

Jeffery, 1995Russo, 1996

cycA-ckk2-p27 X-ray: 2.3 Å p27 E. coli (soluble) Russo, 1996No strcuture GST-cdk4; cdk4 Baculo/insect cellscdk4 (mimic cdk2) X-ray cdk2, engineered

cdk4 pocketBaculo/insect cells Ikuta, 2001

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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

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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)

r pharmaceuticals d. ambrosius; slide 1 proteine/ramc-presentation-9-01 various strategies used to...

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