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Various Strategies Used
to Obtain Proteins for
Crystallization and Biostructural Studies
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Dorothee Ambrosius, R. Engh, F. Hesse,
M. Lanzendörfer, S. Palme, P. Rüger
Roche Pharmaceutical Research, Penzberg
D. Ambrosius; slide 1
Proteine/RAMC-Presentation-9-01
Protein Classes
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transporter (albumin)
immuno-globulin
enzymes, enzyme-inhibitors
coagulation factors, lipoproteins
 protein characteristics/
stability
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often monomeric proteins
contain disulfide bridges
protease resistant
stable fold
D. Ambrosius; slide 2
intracellular proteins
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extracellular proteins
 plasma protein concentration:
 70 mg/ml
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 cytoplasma and organelles:
 300-800 mg/ml
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multi-enzyme complexes
enzyme cascades
transcription complexes
focal adhesion/integrins
cytoskeleton, heat-shock proteins
 protein characteristics/stability
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often multimeric complexes
no disulfide bridges
very labile proteins; short half-life
require stabilization: interaction with
other proteins
Proteine/RAMC-Presentation-9-01
Protein Sources/Expression Systems
Expression system Advantages
Examples
Structure
E. coli
 rapid cloning/ expression
 soluble
 high yield
 inclusion bodies  isotope labeling possible
G-CSF; IBs
PEX, IBs
MIA, IBs
IL-16, soluble
MDM2, IBs
PKA, soluble
NMR
X-ray
NMR
NMR
X-ray, NMR
X-ray
Baculo/
Insect cells
 expression of active protein
 modifications
RTS: E. coli
see talk & poster
 parallel expression
 high throughput proteomics J. Stracke
D. Ambrosius; slide 3
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most Tyr kinases X-ray/NMR
(RTK: IRK,c-met,
SRC, LCK, etc.)
Ser/Tyr kinases X-ray/NMR
e.g. cdks, cAPK
Proteine/RAMC-Presentation-9-01
Biological Function of Cytokines
G-CSF
Neutrophils
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Source: Herrmann/Lederle
D. Ambrosius; slide 4
Proteine/RAMC-Presentation-9-01
 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
 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
D. Ambrosius; slide 5
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
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Development Goals for Recombinant Human G-CSF
Proteine/RAMC-Presentation-9-01
Strategy: Development of Recombinant Human G-CSF

Fusion Peptide
Human G-CSF
Fusion Peptide
Protease
rhG-CSF
 high level expression
 specific
 low production costs
 improved refolding
 efficient
 without N-terminal Met
 efficient separation of cleaved
and uncleaved protein
 recombinant
 equal potency/efficiency
 consistent quality
 consistent quality
 optimized cleavage site
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 improved quality
Genetic engineering of an economic downstream process
D. Ambrosius; slide 6
Proteine/RAMC-Presentation-9-01
Optimization of rhG-CSF Fusion Proteins
Expression
Cleavage
(%)
Renaturation
(%)
Met G-CSF
100
-
10
Met-Thr-Pro-Leu  G-CSF
30
++
20
Met-Thr-Pro-Leu-His-His  G-CSF
100
++
20
Met-Thr-Pro-Leu-Lys-Lys  G-CSF
100
+
50
Met-Thr-Pro-Leu-Glu-Glu-Gly  G-CSF
25
+++
90
Met-Thr-Pro-Leu-Glu-Glu-Gly-Thr-Pro-Leu  G-CSF
10
++
80
Met-Lys-Ala-Lys-Arg-Phe-Lys-Lys-His  G-CSF
100
+++
80
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Fusion Peptide
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Cleavage Site (Pro-Arg-Pro-Pro)
Source: EP 92102864.3 ; DE 4104580
D. Ambrosius; slide 7
Proteine/RAMC-Presentation-9-01
Refolding Kinetics of rhG-CSF Fusion Protein
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
Pellet
SN
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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
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denat.
Source: EP 92102864.3 ; DE 4104580
D. Ambrosius; slide 8
Proteine/RAMC-Presentation-9-01
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h
active p53
latent p53
activation
accumulation
stress factors
or oncogenic proteins
mdm2
negative feedback loop !!
D. Ambrosius; slide 9
cell type
level of p53
extent of DNA damage
genetic background
cell cycle arrest:
repair defective genes
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Role of p53 in cell cycle control:“guardian of the genome”
apoptosis:
kill harmful
deregulated cells
Proteine/RAMC-Presentation-9-01
The MDM2 oncoprotein is a cellular inhibitor of the p53 tumor
suppressor.
Goal:
Improvement of biophysical properties of HDM2
(human MDM2) by “crystal engineering”
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Pharmaceuticals
Engineering of MDM2 for biostructural purposes
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
D. Ambrosius; slide 10
Proteine/RAMC-Presentation-9-01
Structure of MDM2/p53-peptide complex
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Figures taken from Kussie et al., Science 274 (1996) 948.
Resolution X-ray structures:
human MDM2/p53: 2.6 Å
Xenopus MDM2/p53: 2.3 Å
D. Ambrosius; slide 11
17-29
p53
mdm2
26-108
Proteine/RAMC-Presentation-9-01
MDM2 variants created by protein engineering
1
26
108
125
185
240
300
330
350
440
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
XDM2 (21-105) LHI
D. Ambrosius; slide 12
I50L
P92H
L95I
491
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human MDM2
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NMR, X-ray
X-ray
Proteine/RAMC-Presentation-9-01
Human MDM2: Yields & Upscale
15N-labeled
(minimal medium)
non-labeled (LB)
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
Renaturation (~25%)
0.2 g
MDM2 (Purification)
0.16 g
3.6 g
Final product
0.1 g
2.2 g
D. Ambrosius; slide 13
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Step
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18 g
4.5 g
Proteine/RAMC-Presentation-9-01
Crystals of hXDM/peptide
Patience might be rewarded
hXDM2/phage-peptide
Conditions: 0.1 M MES pH 6.2, 4.0 M NaOOCH
3 days after micro seeding at 13 °C
D. Ambrosius; slide 14
hXDM2/p53 peptide
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Some crystals comply with
corporate identity rules
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4 months at 4 °C
Proteine/RAMC-Presentation-9-01
Protein Kinase Families (incomplete list)
cAPK: cAMP dependent protein kinase
cdks: Cyclin dependent kinase
MAPK: Mitogen activated protein kinase
MLCK: Myosine light chain kinase
CK: Casein kinase
PhK: Phosphorylase kinase (tetramer: , , , )
CaMK: Calcium/calmodulin dependen kinase
Subfamilies/Structures
PKA, PKB, PKC
cdk2, cdk4, cdk6
Erk, Erk2, Jnk, p38(,,)
Twitchin, Titin
Ck-1, Ck-2
PhK
CaMK
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I: Ser/Thr-Kinase Families
Ia: Non Receptor Ser/Thr-Kinase familiy
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Ib: Receptor Ser/Thr-Kinase family
TGF1-R Kinase
II: Tyr-Kinase Families
IIa: Non receptor Tyr-Kinase family
TGF1-ßR
Subfamilies/Structures
SRC-family
SRC, c-SRC, CSK, HCK
LCK: humam lymphocyte kinase:
LCK, c-Abl
IIb: Receptor Tyr-Kinase family
EGFR-family:
InsR-family
PDGFR-, CSFR-, Met-, Ron-familiy,
EphA1….EphB1, Trk A, B, C, etc.
D. Ambrosius; slide 15
EGFR, ErbB2-4
IRK, IGF1R, IRR
FGF1-R, VEGFR-K
Proteine/RAMC-Presentation-9-01
Further details for crystallization see poster of Ch. Breitenlechner
D. Ambrosius; slide 16
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Pharmaceuticals
PKA: 2 Å X-ray Structure
Proteine/RAMC-Presentation-9-01
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
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Pharmaceuticals
PKA: cyclic AMP Dependent Protein Kinase
Ideal model: Ser/Thr protein kinase inhibitor studies
generation of other Ser/The kinase (e.g. PKB, Aurora) structures
D. Ambrosius; slide 17
Proteine/RAMC-Presentation-9-01
Major Components of the Cell Cycle Machinery
CDC2
INK4
M
cyclin B
Mitosis
Cell
Cycle
G2
CDK4/6
G1
cyclin D
CDC2
DNA Replication
cyc. A/B
S
CDK2
cyclin A
D. Ambrosius; slide 18
Kip/
Cip
CDK2
cyclin E
Kip/
Cip
 mitogen induced progression
through the cell cycle requires
timely controlled activation of
different cyclin-dependent
kinases (CDKs)
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G0
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 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
Proteine/RAMC-Presentation-9-01
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Pharmaceuticals
Cyclin Dependent Kinases: CDK2 and CDK4/6
N. Pavletich, JMB 287, 821-828, 1999
D. Ambrosius; slide 19
Proteine/RAMC-Presentation-9-01
Structural investigations of cdks (incomplete list)
Method
p16
p16
p18
Expression system
Reference
Folding studies p16
NMR
GST-p16
NMR
GST-p18
E. coli (IBs)
E. coli (soluble)
E. coli (soluble)
Tang, 1999
Byeon, 1998
Yuan, 1999
p18
p19
p19/cdk6, p16/cdk6
X-ray: 1.95 Å
NMR
X-ray: 2.8 Å
X-ray: 3.4 Å
p18
p19
cdk6
GST-p19/p16
BL21 (soluble)
E. coli (IBs)
Baculo/insect cells
E. coli (soluble)
Venkataramani, 1998
Baumgartner, 1999
Russo, 1998
p19/cdk6
X-ray: 1.9 Å
p19
GST-cdk6
GST-cycK
GST-p18
cdk6
cdk2
cycA:
p27
GST-cdk4; cdk4
cdk2, engineered
cdk4 pocket
E. coli (soluble)
Baculo/insect cells
E. coli (soluble)
E. coli (soluble)
Baculo/insect cells
Baculo/insect cells
E.coli (soluble)
E. coli (soluble)
Baculo/insect cells
Baculo/insect cells
p18/cdk6/cycK
X-ray: 2.9 Å
cycA-cdk2
cycA-ATPS-cdk2
cycA-ckk2-p27
No strcuture
cdk4 (mimic cdk2)
X-ray: 2.3 Å
X-ray: 2.6 Å
X-ray: 2.3 Å
D. Ambrosius; slide 20
X-ray
Protein
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Structure
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Brotherton, 1998
Jeffrey, 2000
Jeffery, 1995
Russo, 1996
Russo, 1996
Ikuta, 2001
Proteine/RAMC-Presentation-9-01
 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
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Pharmaceuticals
Summary
 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
D. Ambrosius; slide 21
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:
D. Ambrosius; slide 22
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S. Stammler, M. Leidenberger, U. Michaelis,
T. Zink (MPI), T. Holak (MPI)
Proteine/RAMC-Presentation-9-01