Affinity Chromatography IEX – SEC Amylose, Strep, improved ...€¦ · Wyatt Dyna Pro Plate...

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


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


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



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 NaCl5% Glycin

5% Mannitol

150mM NaCl5% Glycin

5% Mannitol

300mM NaCl5% Glycin

5% Mannitol

TrispH 8,0

20mM NaCl

150mM NaCl

300mM NaCl




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



ANSECÄktaBasic, SuperdexPC

BlueNative

MALS

Acknowledgements

Stephan

Uebel

Claudia Franke

Judith ScholzLissy WeyherEvi Stieger

Affinity Chromatography IEX – SEC Amylose, Strep, improved ...€¦ · Wyatt Dyna Pro Plate...

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Transcript of Affinity Chromatography IEX – SEC Amylose, Strep, improved ...€¦ · Wyatt Dyna Pro Plate...