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Protein Purification PEPC5: 28th Aug - 5th Sep 2006 Young-Hwa Song, EMBL-Hamburg

Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent

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Page 1: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent

Protein Purification

PEPC5: 28th Aug - 5th Sep 2006Young-Hwa Song, EMBL-Hamburg

Page 2: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent

Fermentation using the autoinducing medium ZYP-5052:

- developed by Studier (BNL)

- Phosphate buffered “rich medium”, 50 mM pH6.7

- Contains glucose (0.05%), glycerol (0.5%), α-lactose (0.2%) and

1 mM MgSO4

- Glucose and Glycerol support bacterial growth to high density up to 20 OD

- Glucose suppresses lactose permease, in pET-system the T7-polymeraseexpression

- after depletion of glucose lactose acts as inducer

Studier’s autoinducing media

Page 3: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent

“It is not unusual to find that the high densities of induced cultures attained inZYP-5052 provide as much as ten times the amount of target protein as thesame volume of culture induced with IPTG in the conventional way.”

autoinducing conventional LB

Page 4: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent

(1) General thoughts about protein purificationWhy do we need to purify? Value and things to consider.

(2) Steps of purification(3) Introduction to liquid chromatography(4) Automated chromatography

Page 5: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent

Value of protein purification

Only with purified protein one can study the following:

- correlation of a certain biological activity to a particular protein- can be sequenced either Edmund degradation or MS-Probe the gene and isolate the gene, that encodes the protein of interest

Study structural and functional relationship by X-ray crystallography or/and NMR

Page 6: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent

Before starting with purification find outHow to

monitor the purification with an assay

The assays can be as diverse as you are creative:- SDS-band intensity is not sufficient

- enzymatic activity (NADPH…) if it is an enzyme- the ability of binding of certain ligand

- a shift in a gel after DNA or RNA binding- monodispersity with correlation of its molecular weight

- the ability to stimulate cell death of cell proliferation- the ability to stimulate cell differentiation

……..BUT important: it should be a measurable quantity

Page 7: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent

SampleCrudeextract

Separationtechnique

Fractionation

Purification is a multi-step procedure

Is there activity?Set asideNo Combine

Fractions

yes Monitor purity

Assay total protein

Assay enzyme activity

Pure?

Prepare for analytical technique

yes

No

Repeat with anotherseparation

technique until pure

Page 8: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent

First Step of Protein Purification in Practice

Sample extraction: question of solubility of theexpressed targets in a proper conditions

- select proper buffer: pH, salt, detergents, Chaotropic agents, metalions, reducing agent,

- Add Dnase, protease inhibition agents

Commonly used lysis buffers for His6-tagged protein:50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 20-50 mM Imidazol,(1-2 M Urea or othe chaotropic agents)

Lysis buffer for GST-tagged protein:PBS, pH 7.3: 10 mM Pi-buffer, 140 mM NaCl

Usually done by sonication or FrenchPress lysis followed by aHigh-spin centrifugation (~ 50.000xg rcf)

Monitor the extraction: SDS, activity assay if available.

Page 9: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent

Liquid Chromatography

Liquid flow

Liquid flow

Time 1 2 3 4 5

Separation according to: -molecular weight/ size-charge-hydrophobicity-affinity

Sample containing proteins or peptides

Page 10: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent

Proteins are Macro Poly-Ions

Baker et al. 2001Song et al. 2001

Page 11: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent

Hydrophobic interactionchromatography (HIC)

Reverse phase chromatography (RPC)

Hydrophobicity

Affinity chromatographybinding to small molecules

Ion exhange chromatographyIsoelectric point (charge)

Size-exclusion chromotographySize / shape

Precipitation with AS

(salting out)

Solubility

MethodsProperty

2nd step: separation techniques

Page 12: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent

Affinity ChromatographySolvent flow

Bead with covalentlyAttached substrate(glutathione, Ni-NTA, etc)

Bound enzyme molecule(GST, His6)

Other proteins withoutTag---> pass through

Page 13: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent

Ion ExchangeSolvent flow

Positively chargedbeads

Bound negativelyCharged molecules

Free positivelyCharged molecules

Anion exchage matrix: DEAE-spharose, Mono-Q, SOURCE-Q, RESOURCE-Q, etcKation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc

Page 14: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent

Gel FiltrationSolvent flow

Porous beads

Retarded small molecules

Unretarded large molecules

Page 15: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent

Properties of gel filtration media

(1) Sephacryl HR: xlinked allyl dextran with bisacrylamde

stable pH 3-11, 8M Urea, 0.5M NaOH etc

(2) Superdex: a composite gel consisting of highly xlinked porous agarose and covalently bonded dextran.stable pH 3-12, 8M Urea, 0.5M NaOH, 0.5M HCl etc

(3) Superose: xlinked porous agarose beads

(4) Sephadex: xlinked dextran

(5) Sepharose: bead-formed gel prepared from agarose, melts above 40°C

Page 16: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent

Selection and combinatioinof purification technique

Minimize sample handlingMinmize number of stepsuse different techniques at each step

Every technique offers a balance Between resolution, speed, capacity and recovery

Page 17: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent

Purity

Step

Capture

Intermediatepurification

Polishing

Isolate product,concentrate, stabilize

Remove bulkimpurities

Achieve final purity.Remove trace impurities,structural variants,aggregates, viruses, etc.

Three Phase Strategy: considerations whendeveloping the purification scheme

Page 18: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent

Suitability estimation of purification techniquesConcerning the three phase purification strategy

Page 19: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent

Always Limit the Number of StepsMaximize the Yield at Each Step

Number of steps

Yield (%)

95% / step

90% / step

85% / step

80% / step75% / step

0

20

40

60

80

100

1 2 3 4 5 6 7 8

20% overallyield!

Page 20: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent

What are the requirements of proteinqualities in structural biology

• >90 % purity of the proteincriteria of purity: constant specific activity or homogenity

• automation

• mgs of protein

• AND if you have a HTP xtal-facility

• a throughput of >10 different tagged proteins perday

Page 21: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent

58%

66%

Page 22: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent

Automated purification using Akta-explorer with 3D-kit

- His- &GST tagged targets

- Multiple samples (up to 6)

- Up to 3 steps

Page 23: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent
Page 24: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent

Automated purification of six samples using 3D-kit

Page 25: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent
Page 26: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent
Page 27: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent

Automated purification using Äkta-explorer with 3D

sample loading sample 1 sample 2

sample 3 sample 4 sample 5

Page 28: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent

Boi2 Hse1 NBP2 RVS YHH6

S P S P S P S P S PMw

Bo

i2

Hse

1

NB

P2

RV

S

YH

H6

Mw

Expression and solubility

Two step purificationIMAC and Gelfiltration

Using Akta-3D

16% tricine-SDS

6

3

14

6

3

14

28

Page 29: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent

Removal of His-tags

Mw Boi2 NBP2 RVS YHH6

- + - + - + - + - +

Hse1

6

3

Mw Boi2 NBP2 RVS YHH6Hse1

Tev-cleavage 2nd Imac-step

Page 30: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent

ÄKTAxpress: automated HTP-purificationsabout 50 proteins / week

Page 31: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent

Supported Protocols usingÄKTAxpress™

AC - DSAC - GFAC - DS - IEXAC - DS - IEX - DSAC - DS - IEX - GF

DS or GF only

Automated multi step protocolswith or without tag removal

Manual loading

AC = Affinity ChromatographyDS = DesaltingIEX = Ion Exchange ChromatographyGF = Gel Filtration (size exclusion)

Page 32: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent
Page 33: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent
Page 34: Protein Purification - EMBL Hamburg · First Step of Protein Purification in Practice ... Kation exchange matrix: SP-sepharos, Mono-S, SOURCE-S, RESOURCE-S, etc. Gel Filtration Solvent

Acknowledgements

-Thanks to the co-organizers-All wetlab members of the EMBL-HH-All the helpers, Christian and Lei

-GE Healthcarefor AktaXpress

-Thanks to your attention.