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Services and MaterialsP4 at the MPIBPurpose, Problems, Procedures, Perspectives
Purpose
Problems
Procedures
and Perspectives
Affinity
Chromatography
–
IEX –
SEC
Amylose, Strep, improved
IMAC tags
IEX -
HIC –
Screening, automation
on Äkta
Avant
Refolding
screens
DoE
Buffer -
Stability
Screening: Thermofluo, CD and DLS
Quality
control
Services and MaterialsP4 at the MPIBPurpose, Problems, Procedures, Perspectives
Purpose
currently
45 proteins
/ yr
1 week
to 1 year
projects
> 95% intracellular, no membrane
proteins
, few
protein
complexes
DNA, RNA-
binding
proteins, structural
proteins, proteases, chemokines
etc
Interaction studies
(Biacore, ITC), crystallization, immunization
etc
Problems
Soluble
aggregates
Sticky
proteins
Degradation (copurification
of full
length
+ degradation
products)
LPS removal
Procedures: Improved IMAC tags
Peptide*Ni-NTA rt [min]
CoTalon rt [min]
DHLIHNVHKEEHAHAH 10,0 6,4
HHHHHHGSGS 19,1 13,0
HHHHHHHHGS 25,4 20,7
HIHNLDCPCDC 6,3 4,0
HIHNLDHPDH 8,9 6,0
HITGLDCPDC 2,7 1,3
DHLIHNVHAH 9,0 6,0
HHHHHHHHHHGS 23,4 23,2
HPHHGGHPHHGS 14,5 10,5
HNHRYGHGSHGS 13,8 10,0
AHEFGHALGLDHS 5,0 1,4
VHELHGHALGLEHS 4,2 1,3
HDHGSDSLHGHSHGS 10,5 6,7
HHHSGEGQGAGHGHSHGS 12,7 7,9
HDHGLDHGSH 8,4 5,4
His6
versus
His8
versus
His10
and
binding
motifs
of metal binding
proteins
HPLC retention
times
of peptides
bound
to Ni and Co in imidazole
gradient
His10
and His8
> His6
Recovery
of His6 or
His8
or
His10
eGPF
spiked
into
BL21 (DE3) lysate
by
IMAC
H6
H8
H10
H6
H8
H10
H6
H8
H10
H10
150mM 200mM 250mM 300mMImidazole
eGFP
recovery
eGFP
purity
GFP recovery
0
50
100
150
200
250
300
His6 His8 His10
ng/
µl
150 mM200 mM250 mM300 mM
GFP purity
0
20
40
60
80
100
120
His6 His8 His10
%
150 mM200 mM250 mM300 mM
His6 His8 His10
His6 His8 His10
100 µg purified
His6
, His8
or
His10
–Sumo3eGFP was spiked
into
1 ml BL21(DE3) lysate
and recovered
on Ni beads
Procedures: Improved IMAC tags
His6
-POI
C100
C150
G100
G150
G100
M100
S50
His10
-POI
C150
C200
G150
M100
G200
M100
S100
S100
C His60 Ni Superflow (Clontech)
G Ni High Performance (GE)
M Protino Ni NTA (Macherey Nagel)
S His Select (Sigma)
20kDa
Procedures: Improved IMAC tags
IEX-
HIC Screening
IEX-
HIC Screening
Äkta
Avant: Materials (surface
+ pores) x pH
(buffers)
Material Matrix Accessibility
for
large proteins
Source30SPolystyrene
Divinylbenzene
30µm
low
Sepharose
HP6% cross linked
agarose34 µm
low
Sepharose
HP-XL
6% cross linked
agarose
+ dextran
linker
45-165µm
high
Capto
ImpResHigh-flow
agarose36-44µm
high
Perspective: Higher
degree
of automationfor
screening
column
screens sample
throughput10ml lysate
on 0,2ml Ni beads buffer
screens
on SEC
Thermofluo
Scenario 1
G233
Thermofluo
Scenario 1
Size
Exlusion
Analytical
Ultracentrifugation
Total Mass
Thermofluo
Scenario 2
N270
Thermofluo
Scenario 2
Size
Exlusion
–
MALS (Wyatt Treos)
Analytical
Ultracentrifugation
Molar Mass vs. volume11 N270 175mgmL 10uL Super200 12 N270 175mgmL 50uL Super200 13 N270 175mgmL 250uL Super200
volume (mL)6.0 8.0 10.0 12.0 14.0 16.0 18.0
Mol
ar M
ass
(g/m
ol)
51.0x10
61.0x10
71.0x10
81.0x10
dRI
250 µL N270Mw peak 1 : 6.394×104 (±1.202%) g/molMw peak 2 : 8.300×104 (±0.369%) g/molRh peak 2 : 4.6 (±3.4%) nmMw peak 3 : 6.144×105 (±0.233%) g/molRh peak 3 : 10.2 (±2.2%)Recovery : 96.4 %
Thermofluo
Scenario 3
> protein
is
aggregated
= high fluorescence, no melting
curve
> but
high fluorescence
= aggregated
protein??
Sypro
Orange staining
as indicator
of aggregation?
Journal of Pharmaceutical Science, 2009
Current
project
(structure
protein)
Thermofluo
Scenario 3
Hr helix regular, Hd helix distorted
Sr strand ß, St strand distorted
Trn turns
Unrd unordered
This
protein
has helical
structure
but
starts
aggregating
at 20°C !!!
CD spectrum
at 4°C CD melting
curve
Thermofluo
vs
CD Lysozyme
Thermofluo
CD
Tris
pH8,020mM NaCl
75,8°C
NaAc
pH4,520mM NaCl
78,4°C
Tris
pH8,020mM NaCl
70°C
NaAc
pH4,520mM NaCl
74°C
real Tm, no aggregation
pH8,0
pH4,5
pH8,0pH4,5
pH8,0
Thermofluo
vs
CD Lysozyme
Tris
pH8,020mM NaCl
75,8°C
NaAc
pH4,520mM NaCl
78,4°C
Tris
pH8,020mM NaCl
70°C
NaAc
pH4,520mM NaCl
74°C
real Tm, no aggregation
pH8,0
pH4,5
pH8,0pH4,5
pH4,5
Thermofluo
CD
Thermofluo
vs
CD N270
NaAc
pH4,520mM NaCl
Onsetagg
< 40°C
Tris
pH8,020mM NaCl
Onsetagg
> 45°C
NaAc
pH4,520mM NaCl
44,6°C
Tris
pH8,020mM NaCl
50°C
no Tm, onset
of aggregation
+ precipitation
pH8,0
pH4,5
pH8,0
pH4,5
pH8,0pH4,5
pH8,0
pH4,5
Thermofluo
CD
Thermofluo
vs
CD Ta protein
NaAc
pH4,520mM NaCl
Onsetagg
50°C
Tris
pH8,020mM NaCl
Onsetagg
> 80°C
NaAc
pH4,520mM NaCl
59°C
Tris
pH8,020mM NaCl
87°C
no Tm, onset
of aggregation
+ precipitation?
pH8,0pH4,5
pH8,0
pH4,5
pH8,0
pH4,5
pH8,0
pH4,5
Thermofluo
CD
Thermofluo
vs
CD ODC
ß
–sheet , Dimer
PBS 49°C ???
Tris
pH8,0300mM NaCl5% GlyMann
80°C ???
no Tm, onset
of aggregation
+ precipitation
PBS no precipitation
at 90°C
Tris
pH8,0300mM NaCl5% GlyMann
Onsetagg
70°C
Thermofluo
CD
Tris
PBS
Tris
PBS
Tris
PBS
Tris
PBS
DLS as 2nd HTP method ?
Wyatt Dyna
Pro Plate Reader
Melting curves 22 °C (cooling optional) -
80°C in 15h on 384 well
Lysozyme, N270, Ta
in 12 buffers
Na-Acetate pH 4,5
20mM NaCl
150mM NaCl
300mM NaCl
20mM NaCl10% Glycerol
150mM NaCl10% Glycerol
300mM NaCl10% Glycerol
20mM NaCl5% Glycin
5% Mannitol
150mM NaCl5% Glycin
5% Mannitol
300mM NaCl5% Glycin
5% Mannitol
Na-Phosphate
pH 7,0
20mM NaCl
150mM NaCl
300mM NaCl
20mM NaCl10% Glycerol
150mM NaCl10% Glycerol
300mM NaCl10% Glycerol
20mM NaCl5% Glycin
5% Mannitol
150mM NaCl5% Glycin
5% Mannitol
300mM NaCl5% Glycin
5% Mannitol
TrispH 8,0
20mM NaCl
150mM NaCl
300mM NaCl
20mM NaCl10% Glycerol
150mM NaCl10% Glycerol
300mM NaCl10% Glycerol
20mM NaCl5% Glycin
5% Mannitol
150mM NaCl5% Glycin
5% Mannitol
300mM NaCl5% Glycin
5% Mannitol
DLS Lysozyme
1
2
3
20 30 40 50 60 70 80
Rad
ius
(nm
)
Temp (C)
Lyso C1Lyso C2Lyso C3Lyso C4Lyso C5Lyso C6Lyso C7Lyso C8Lyso C9Lyso C10Lyso C11Lyso C12
no aggregation
observed
= CD (Tm, but
no aggregation)
(preference
for
acidic
buffer
indicated
by
Thermofluo
and CD)
56
55
53
50
56
55
53
51
49
48
41
45
TmThermofluo
25.8
24
17.6
20
50
22.9
19.2
19
57
42.4
56.8
30.3
Radius (nm)Sample Tm DLS
pH4,5 20mM 52.3 (?)
pH4,5 300mM 28.9
pH4,5 300mM 10% Gly 32.8
pH4,5 300mM 5% GM 38.3
pH7,0 20mM 48.8
pH7,0 300mM 46.6
pH7,0 300mM 10% Gly 52.2
pH7,0 300mM 5% GM 52
pH8,0 20mM 45.3
pH8,0 300mM 45.6
pH8,0 300mM 10% Gly 50.4
pH8,0 300mM 5% GM 50.4 56
55
53
50
56
55
53
51
49
48
41
45
TmThermofluo
25.8
24
17.6
20
50
22.9
19.2
19
57
42.4
56.8
30.3
Radius (nm)Sample Tm DLS
pH4,5 20mM 52.3 (?)
pH4,5 300mM 28.9
pH4,5 300mM 10% Gly 32.8
pH4,5 300mM 5% GM 38.3
pH7,0 20mM 48.8
pH7,0 300mM 46.6
pH7,0 300mM 10% Gly 52.2
pH7,0 300mM 5% GM 52
pH8,0 20mM 45.3
pH8,0 300mM 45.6
pH8,0 300mM 10% Gly 50.4
pH8,0 300mM 5% GM 50.4
DLS N270
DLS Ta
8.8
77nd
77.3
Radius (nm)Sample Tm DLS Tm Thermofluo
pH4,5 20mM < 22 59
pH4,5 300mM < 22 59pH4,5 300mM 10% Gly < 22 60pH4,5 300mM 5% GM < 22 64pH7,0 20mM 59.1 88pH7,0 300mM 60.1 89
pH7,0 300mM 10% Gly no aggregation 88
pH7,0 300mM 5% GM nd 91pH8,0 20mM 56.6 87pH8,0 300mM 62 88
pH8,0 300mM 10% Gly 69.6 88
pH8,0 300mM 5% GM no aggregation 91
8.8
77nd
77.3
Radius (nm)Sample Tm DLS Tm Thermofluo
pH4,5 20mM < 22 59
pH4,5 300mM < 22 59pH4,5 300mM 10% Gly < 22 60pH4,5 300mM 5% GM < 22 64pH7,0 20mM 59.1 88pH7,0 300mM 60.1 89
pH7,0 300mM 10% Gly no aggregation 88
pH7,0 300mM 5% GM nd 91pH8,0 20mM 56.6 87pH8,0 300mM 62 88
pH8,0 300mM 10% Gly 69.6 88
pH8,0 300mM 5% GM no aggregation 91
= CD
onset
CD 80°C
Summary Thermofluo
so far….
Thermofluo
measures
Tm and aggregation
Data are
better
than
expected, in good agreement
with
CD and DLS
But
unexplained
effects
for
certain
proteins
No
Tm measurement
data
for
aggregated
proteins
Strategy
–
Perspective
First screen
thermofluo
Confirm
2-3 selected
buffers
by
CD –Tm (no HTP !)
Confirm
by
SEC –
MALS
Complement
with
additional method
like
DLS –
Tm
,
(intrinsic
fluorescence
-Tm, Avacta
LS + intrinsic
fluorescence)
Quality control
SILAC, PTMLTQ-Orbitrap
Agilent
Bioanalyzer
CD Jasco715,810
ThermofluorLight Cycler
480 II
AUC Beckman
CoulterSEDFIT, SEDPHAT
DLS-
Tm
ESI-TOFBRUKER microTOF
1,00 10,00 100,00
Radius(nm)
Radius(nm): 7.751 %Pd: 26.2 Mw -R(kDa): 406 %Intensity: 33.4 %Mass: 97.6
Radius(nm): 73.518 %Pd: 36.3 Mw -R(kDa): 78372 %Intensity: 61.3 %Mass: 1.7
Radius(nm): 267.900 %Pd: 14.3 Mw -R(kDa): 1614910 %Intensity: 5.3 %Mass: 0.7
ANSECÄktaBasic, SuperdexPC
BlueNative
MALS
Acknowledgements
Stephan
Uebel
Claudia Franke
Judith ScholzLissy WeyherEvi Stieger