A difficult to produce RNA binding protein. On-column refolding

Ion exchange some applications, etc Effective removing of tightly bound nucleic acids from over-

expressed proteins. On-column refolding

Dr. Mario Lebendiker The Protein Purification Facility

THE HEBREW UNIVERSITY OF JERUSALEM THE WOLFSON CENTRE FOR APPLIED STRUCTURAL BIOLOGY

IBMC-INEB - P4EU WORKSHOP ON PROTEIN CHARACTERIZATION November 11th , 2013 Porto

2

Critical Factors in IEX resolution

Shape and volume of the gradient

Effect of flow rate on resolution

Effect of pH

Effect of different salts

Use of additives (detergents, ligands,

co-factors, etc

Effect of temperature

Column length (volume)

Type of ligand (strong, weak, mixed)

Degree of substitution

Particle size of matrix (efficiency)

Supplier

0

10

20

30

40

50

0 20 40 60 80 100 120

mAU

Elution volume (ml)

M NaCl

0

0,01

0,02

0,03

0 40

1800 cm/h (4.98 ml/min) 900 cm/h (2.49 ml/min) 300 cm/h (0.83 ml/min) 200 cm/h (0.55 ml/min)

0

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0 27

0

0,01

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0 9

Ion exchange some applications: Quality control: change of gelatin conformation with time

Bef 22 24 32 33 34 35 MW 36 37 38 40 42 44 46

MW 5 7 9 10 11 12 13

P38

Ion exchange some applications: Separation of Phosphorilated forms of recombinant kinase

AEIX after IMAC

SEC of last pool

5

Separation of phosphorilated forms of recombinant kinase

Highly resolutive anion

exchange Resource 15Q 29 x 1cm ~23ml column

Ron Diskin et al J. Mol. Biol. (2007) 365, 66–76

Resource15Q23mlB003a001:1_UV1_280nm Resource15Q23mlB003a001:1_UV2_260nm Resource15Q23mlB003a001:1_Conc Resource15Q23mlB003a001:1_Fractions Resource15Q23mlB003a001:1_Logbook

0

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mAU

80 100 120 140 160 180 ml

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

1st run main peak of Affinity column

2nd run: main peak of previous run

Resource15Q23mlB003:1_UV1_280nm Resource15Q23mlB003:1_UV2_260nm Resource15Q23mlB003:1_Conc Resource15Q23mlB003:1_Fractions Resource15Q23mlB003:1_Logbook

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600

mAU

80 100 120 140 160 180 200 ml

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 Waste

Ion exchange some applications: Separation of Glycosilated forms of recombinant secreted

protein from insect cells

10 11 12 14 15 17 18 19 20 21 23 25 28 36

Superdex75an001Del Rolled after IEX Frac 20 to 21 12912:1_UV1_280nm Superdex75an001Del Rolled after IEX Frac 20 to 21 12912:1_UV2_260nm Superdex75an001Del Rolled after IEX Frac 20 to 21 12912:1_Fractions Superdex75an001Del Rolled after IEX Frac 20 to 21 12912:1_Inject Superdex75an001Del Rolled after IEX Frac 20 to 21 12912:1_Logbook

0.0

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mAU

0.0 5.0 10.0 15.0 20.0 25.0 30.0 ml

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Waste

12.9ml

Del Rolled after IEX Frac. 20 to 21 12.9.12

Superdex75an001 Del Rolled after IEX Frac 17 to 18 12912:1_UV1_280nm Superdex75an001 Del Rolled after IEX Frac 17 to 18 12912:1_UV2_260nm Superdex75an001 Del Rolled after IEX Frac 17 to 18 12912:1_Fractions Superdex75an001 Del Rolled after IEX Frac 17 to 18 12912:1_Inject Superdex75an001 Del Rolled after IEX Frac 17 to 18 12912:1_Logbook

0.0

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mAU

0.0 5.0 10.0 15.0 20.0 ml

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Waste

9ml

12.9ml

Del Rolled after IEX frac. 16-17

Resource15Q23mlAir overnight001 Del Rolled from after Ni Frac 3 to 13 11912:1_UV1_280nm Resource15Q23mlAir overnight001 Del Rolled from after Ni Frac 3 to 13 11912:1_UV2_260nm Resource15Q23mlAir overnight001 Del Rolled from after Ni Frac 3 to 13 11912:1_Cond Resource15Q23mlAir overnight001 Del Rolled from after Ni Frac 3 to 13 11912:1_Conc Resource15Q23mlAir overnight001 Del Rolled from after Ni Frac 3 to 13 11912:1_Fractions Resource15Q23mlAir overnight001 Del Rolled from after Ni Frac 3 to 13 11912:1_Logbook

0

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mAU

550 600 650 700 750 ml

F3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 Waste

Del Rolled after Ni and dilu. 11.9.12

Resource 15 Q 30x1cm ~23ml Load protein after Ni column and

dilution X 10 with buffer A (low salt)

Superdex 75 anal. 30x1cm ~23ml

Ion exchange some applications: SEC Replacement by EIX to facilitate scale-up

U.B

.

1 2 3 8 10 11 12 13 15 17

9

Lysa

te

Rab α EGF

was

h

RNase - +

OD260 >OD280

Low binding RNA contamination

72KDa- 56KDa-

36KDa-

28KDa-

17KDa-

Affinity Purification: Low Binding + contamination: RNA and other proteins

Conventional purification steps to remove nucleic acid

Nuclease digestion

Ultracentrifugation steps

Cation exchange or Heparin columns

Precipitation with polyelectrolytes as PEI or Streptomycin

PEI is a positively charged polyelectrolyte (pKa 9.7) with the structural formula (-CH2-NHCH2-)n

RNA-binding proteins are known to have a relatively broad specificity in

binding to RNA

Such proteins may bind to host RNA or DNA during over-expression and co-purify

during the purification process

This tight association of RNA-binding proteins with contaminating cellular nucleic

acid is detrimental to a variety of assays, including RNA-binding analysis, a variety of

spectroscopic studies, and structural analysis.

Challenge 1: removing RNA contamination and improving column binding

Changing expression conditions

High salt concentration, detergents, etc

Precipitation of nucleic acids with PEI

RNase treatment

CEIX before or after Ni

Adding urea

4M Urea

Was

h

Without Urea

Bef

ore

UB

Elu

tio

n

Was

h

Bef

ore

UB

Elu

tio

n

Rapid and Efficient Purification of RNA-Binding Proteins: Application to HIV-1 Rev Marco Marenchino, David W. Armbruster, and Mirko Hennig* Medical University of South Carolina, Protein Expr Purif. 2009 February ; 63(2): 112–119

Refolding Buffer Screening on Column

According to a modification of Sharapova O.A., et al., J. Chromatogr. A (2011), doi:10.1016/j.chroma.2011.05.075

Lyse cells in the presence of Urea

Bind to Ni resin

Wash intensively with buffer in Urea

Aliquot resin in different tubes

Dilute resin ten times with different refolding buffers

Allowed protein to refold during 1 h at constant agitation at 4°C, and then leave ON 4°C.

Elute next day

Check turbidity of the samples. Spin 12500 RPM for 20 min at 4°C. Check PAGE-SDS of supernatant

Keep samples ON 4°C and repeat last step

Check oligomeric state (aggregation) of the relevant samples by analytical GF (no turbidity and more

protein according to the last PAGE-SDS)

Optimize next experiments: type and quantity of additives, sugars, detergents, etc.

Optimize final storage buffer.

Additive

Recommended Initial

Concentration

Recommended

Concentration Range

Sugars and Osmolytes

Glycerol 10% 0-40%

TMAO (trimethylamine N-oxide) 0.5M 0-1 M

Glucose 0.5M 0-2 M

Sucrose 0.5M 0-1 M

Trehalose 0.5M 0-1 M

Ethylene glycol 10% 0-60%

D-Sorbitol 0.5M 0.2-1M

Mannitol 2%

Xylitol 0.5M 0.2-1M

Glycine Betaine 1M

Amino acids and amino acid derivatives

Glycine 250mM 0.5-2%

Arginine L-HCl 125mM 0-2 M

Arginine Ethylester 250mM 0-500 mM

Proline 250mM 0-1 M

Potassium Glutamate 250mM 0-500 mM

Arginine L-HCl + L-glutamic acid (L-Glu) 50mM each

Non-ionic detergents

Nonidet P40 (NP40) or Triton X-100 0.01% 0-1%

Tween 80 or 20 0.1% 0-1%

DDM: n-Dodecyl β-D-maltoside 0.1% 0.01- 0.5%

Brij 56: Polyoxyethylene cetyl ether 0.05%

OG: Octyl glucoside (n-octyl-β-D-glucoside) 0.1% 0.01- 0.5%

Poloxamer 188 (BASF Pluronic® F68)

Zwiterionic detergents

NDSB: Non-detergent Sulfo Betaine 0.5M 0-1M

CHAPS: 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate 0.1% 0.01- 0.5%

Zwittergent 3-14 0.1% 0.001-0.2 %

LDAO: Lauryldimethylamine N-oxide 0.1% 0.01- 0.5%

Ionic detergents

CTAB: cetyltrimethylammonium bromide 0.5%

Sarkosyl : Sodium lauroyl sarcosinate 0.05% 0.01-0.5%

SDS: Sodium dodecyl sulfate Up to 0.1%

Mild chaotrope agents and chaotrope salts

Urea 0.5M 0-2M

Guanidine HCl 0.5M 0-2M

N-Methylurea 250mM up to 2.5M

N-Ethylurea 100mM up to 2M

N-Methylformamide 3-15%

NaI 0.2M 0-0.4 M

CaCl2 10-50 mM 0-0.2M

MgCl2 10-50 mM 0-0.2M

Mild and strong kosmotrope salts

NaCl (weak) 300mM 0-1 M

KCl (weak) 200mM 0-1 M

MgSO4 (strong) 100mM 0-0.4 M

(NH4)2SO4 (strong) 50mM 0-0.2M

Na2SO4 (strong) 500mM 0-0.2M

Cs2SO4 (strong) 50mM 0-0.2M

Potassium citrate 100mM

Citric Acid 50mM

Alcohols, Polyols, Polymers, Polyamines, and others

Ethanol 5-10% Up to 25%

n-Penthanol 1 to 10mM

n-Hexanol 0.1 to 10mM

Cyclohexanol 0.01 to 10mM

Polyethylene glycol (PEG 3350) 0.3-1.5% 0.1-0.4 g/L

Polyvinylpyrrolidone 40 (PVP40) 0.05-4%

Alpha-Cyclodextrin 8-40mM

Beta-Cyclodextrin 1-5mM

Putrescine, spermidine, and spermine 0.1M

Formamide 0.1%

Reducing Agents

β-mercaptoethanol (BME) 2 to 5mM 1 to 10mM

Dithiothreitol (DTT) 1mM 0.1 to 10mM

tris(2-carboxyethyl)phosphine (TCEP) 1 to 5mM 1 to 50mM

Table: Additives Used to Stabilize Folding and to Prevent Aggregation Summary table of different publications (De Bernardez Clark 1999, Voziyan 2000, Goloubinoff 2001, Bondos 2003, Golovanov 2004, Hamada 2009, Shukla 2011, Churion 2012, Leibly 2012) and

from commercial websites (DILYX Biotechnologies OptiSol Protein Solubility Screening Kit Application Manual, HAMPTON: Solubility & Stability Screen)

Most popular parameters in our lab

Control with high salt (0.5M) and 10% glycerol

Non-ionic detergent (0.5%)

Zwittergent (0.5%)

Osmolyte (0.5M)

Urea or GuHCl (1M)

Add Arg only in the elution buffer (0.5M)

UNICORN Method for On-column refolding

Lyse cells with Urea buffer

Incubate 1hr

Batch loading to Ni beads 1.5hr

Spin and build column

AKTA refolding and elution

Wash column with 4M urea buffer 2ml/min (A12)

By-pass column. Wash valves B1 & A11

Wash column and step slow elution

gradient (A11 & B1) Slow ON gradient

4-0 M Urea (A12-B2)

Optional pause

1- Nickel column

2-Sephacryl S-200

3-Superdex 75

3 step purification

Protein yield in purification = 0.25mg

8ml column 500ml

column

320ml column

121112C2ArgPKRscFvSuperdex75prepar520ml001:10_UV1_280nm 121112C2ArgPKRscFvSuperdex75prepar520ml001:10_UV2_260nm 121112C2ArgPKRscFvSuperdex75prepar520ml001:10_Fractions 121112C2ArgPKRscFvSuperdex75prepar520ml001:10_Logbook

-5.0

0.0

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15.0

20.0

25.0

30.0

35.0

mAU

150 200 250 300 ml

F3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62

2- Superdex 75

520ml column Protein yield ~ 1 mg from 0.5L culture

Pool

Pool

Drawbacks Protein lost during further purification

Try to develop an easy and scalable procedure GF cannot be scaled

Ni-NTA Refolding

(Gravity Flow) without AKTA

RESOURCE S + 0.2% Zwittergent

TOYOPEARL MX-Trp-650M + 0.2%

Zwittergent

TOYOPEARL MX-Trp-650M + 0.5%

Tween

RESOURCE S + 0.5% Tween 80

RESOURCE S 8ml+ 0.5% Tween

Anal. Gel Filtration

19

Critical Factors in IEX resolution

Shape and volume of the gradient

Effect of flow rate on resolution

Effect of pH

Effect of different salts

Use of additives (detergents, ligands,

co-factors, etc

Effect of temperature

Column length (volume)

Type of ligand (strong, weak, mixed)

Degree of substitution

Particle size of matrix (efficiency)

Supplier

0

10

20

30

40

50

0 20 40 60 80 100 120

mAU

Elution volume (ml)

M NaCl

0

0,01

0,02

0,03

0 40

1800 cm/h (4.98 ml/min) 900 cm/h (2.49 ml/min) 300 cm/h (0.83 ml/min) 200 cm/h (0.55 ml/min)

0

0,01

0,02

0,03

0 4,5

0

0,01

0,02

0,03

0 27

0

0,01

0,02

0,03

0 9

Ni-NTA

Refolding (Gradient Refolding -

AKTA)

FractoGel + 0.5% Tween 80

RESOURCE S + 0.5% Tween 80

FractoGel+ 0.5% Tween 80

Gel Filtration

+BSA

Gel Filtration

RESOURCE S 8ml + 0.5% Tween 80

Batch binding to Ni-

NTA Refolding + 0.5%

Tween 80 (Gradient Refolding -

AKTA)

Load on FractoGel + 0.05% Tween 80

FractoGel + 0.001% Tween 80

Anal. Gel Filtration

Direct loading on

wide Ni-NTA column Refolding + 0.5%

Tween 80 (Gradient Refolding -

AKTA)

FractoGel + 0.01% Tween 80

FractoGel + 0.001% Tween 80 Column

optimization

CEIX optimization

Large column and mild gradient

131024C2ArgPKRscFvSuperose12anal014:10_UV3_220nm 131024C2ArgPKRscFvSuperose12anal014:10_Fractions 131024C2ArgPKRscFvSuperose12anal014:10_Inject 131024C2ArgPKRscFvSuperose12anal014:10_Logbook

-5.0

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mAU

4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 ml

F3 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Troubleshooting: High viscosity before loading: difficult to filter.

DNA??

Disrupt cells without urea and high DNase, and add urea immediately after lysis and before spin and filter

Eliminate protease sensitive sites from construct