ABRF 2009: Optimization and Application ofOptimization and Application of

Existing and Emerging BiotechnologiesBiotechnologies

February 7-10, 2009 – Memphis, Tennessee

SATELLITE EDUCATIONAL WORKSHOP PROGRAMWORKSHOP PROGRAM

(sw3) Recombinant Protein Laboratory

BASICS AND TIPS OF AFFINITY PURIFICATION

Bacterial cell lysis for purification

Introduction to metal chelation affinity chromatography

Benchtop columns vs AKTABenchtop columns vs AKTA

Standard Operating Procedures used in the St Jude PPF

Details of experimental work

Key Point: All Proteins are Different 

Size

Behavior ChargeHydro h bi it Behavior Chargephobicity

ActivityActivity

A Brief Word About Cell Lysis

Microfluidizer(or French Press) Detergent based Sonication(or French Press)

• High initial outlay for equipment

• No initial outlay• Expensive recurring 

• Popular, as equipment seems 

• Efficient and reproducible

• Works for bacteria, i t ll d

costs, especially on large scale

• Added detergents d t b

easily obtained• Needs close monitoringC tinsect cells and 

yeast(?)• Recommended for large volumes

need to be removed

• Efficiency needs to be optimized based

• Can generate localized heating

• Needs optimizing based on batch sizelarge volumes be optimized based 

on scale• Recommended for “quick and dirty” 

based on batch size• Inefficient• Not recommended (unless you can’t 

extractions( yget a fluidizer)

Requirements for Purification in a lFacility Setting

• Work for a variety of proteinsEasy

• Day‐to‐dayReproducible

• Protein‐to‐proteinReproducible

• Small scale tests to >100 mg prepsScalable

“Conventional” Purification StrategyConventional  Purification Strategy

• Use different property ofUse different property of the POI in subsequent steps– Charge

Polishing

Charge– Size– Hydrophobicity– Isoelectric point– Isoelectric point– Biological affinity

• Need to detect low levels (biological assay?)

Intermediate

(biological assay?)• Multiple intermediate 

steps might be needed

Capture

Affinity (His6‐tag) Purification StrategyAffinity (His6 tag) Purification Strategy

• Engineered affinity for g ynickel

• High selectivity and loading of affinity resin

Polishing

loading of affinity resin provides great purification in one step

• His‐tag allows easy detection

• Fewer (if any)Capture

Intermediate

• Fewer (if any) intermediate and polishing steps required

Capture

Metal Chelation Affinity h h ( )Chromatography (MCAC)

• Histidine forms stable complex with nickel inHistidine forms stable complex with nickel in near‐neutral aqueous conditions

Source: QIAexpressionist

Making Proteins Bind NickelMaking Proteins Bind Nickel• Some proteins naturally chelate metal and will bind to nickelbind to nickel

• A “His‐patch” can be engineered into the 3‐D surface of the protein

• Add a polyhistidine tag– Can be at N‐ or C‐terminalWorks in native and denaturing conditions– Works in native and denaturing conditions

– Usually 6 to 10 His long– Can use consecutive His tagsg– Can be used in conjunction with other tags (TAP‐Tag)– Use a short spacer between tag and POIWid i t f i l t il bl– Wide variety of commercial vectors available

Different formats availableDifferent formats available

• Batch media– Gravity‐flow columns– Pack own FPLC columnsP k d l• Prepacked columns– Different vendors have different connections

• Spin columns• Spin columns– Small scale tests

• Magnetic beadsg• Different Suppliers

– GE, Qiagen, Sigma, 

Different Metals can be UsedDifferent Metals can be Used

Qiagen NTA Superflow columns charged with metals shown

Reagents Compatible with Ni‐NTA MediaReagents Compatible with Ni NTA Media

6 M guanidine HCl 8 M urea 50% glycerol 20% ethanol

2% Triton X‐00 2 M NaCl 2% Tween 20 4 MMgCl2100 2 M NaCl 2% Tween 20 4 M MgCl2

1% CHAPS 5 mM CaCl2 20 mM β‐ME <20 mMimidazole

Basic Protocol For MCAC

Make and clear lysate

Apply to resin

Wash unbound material

Elute POI

TipsTips

•Minimize space for non‐specific binding

Use as small a column as  specific bindingpossible

• Ensure all of background t i h bW h ll proteins have been 

removedWash well

Example: A Well Behaved ProteinExample: A Well Behaved Protein

Example: A Not‐So‐Well‐Behaved Proteinp

Troubleshooting His‐tag PurificationsP bl P ibl S tiProblem Possible reasons Suggestions

Gene for POI not in vectorProtein does not have a his‐tagHis‐tag hidden

Check/sequence plasmidReclone

No POI in eluate

His‐tag hidden

Protein not expressedProtein not in fraction applied

Perform anti‐His Western on the load

Check all fractionsPOI flowed  through column

Check all fractionsCheck buffers, cloning

Wash insufficientReapply to column, wash longer

POI elutes, but notclean

longer

Buffers wrong Remake buffer B

Amount of POI too lowIncrease expressionUse smaller columnUse smaller column

POI very sticky Adjust NaCl, glycerol in buffers

POI l t iBuffers wrong Remake Buffer B

POI elutes in wrong place His‐tag partially hidden

Adjust gradientReclone with tag in different place

Benchtop or SystemBenchtop or System

Cheap Automated

AFlow A

KTA

Gra

vity

G

Benchtop or SystemBenchtop or System

Cheap

Automated

Cheap

AFlow A

KTA

Gra

vity

G

SOP from the St Jude PPFSOP from the St Jude PPF

• Nickel Column [Capture]Nickel Column [Capture]– High capacity AND high selectivity affinity step

• Gel filtration on prep S 200 [I di /P li hi ]• Gel filtration on prep S‐200 [Intermediate/Polishing]

– Adds purification based on higher order structure of proteinof protein

– Use as a buffer exchange step

h ( ) f d d• Ion exchange (IEX) if needed [Polishing]

– Uses charged state of protein

Cleaning up with Gel FiltrationCleaning up with Gel Filtration

Variations to the SOP #1Variations to the SOP #1

• Cleave tag in elution buffer from Ni2+‐column– Ideally, use homemade His‐TEV

• Reapply to column: – POI flows throughPOI flows through

– Tag, contaminants, TEV bind

Variations to the SOP #2Variations to the SOP #2

• Different pore size Superdex columnsDifferent pore size Superdex columns– Fine tune size exclusion based on POI/contaminants

– Limited purification optionsLimited purification options

• Add different polishing stepAdd different polishing step– Hydrophobic interaction (Phenyl, butyl, octyl‐sepharose)p )

– Multimodal (Capto MMC from GE) [HIC and cation]

– Chromatofocusing (MonoP Column)

Variations to the SOP #3Variations to the SOP #3

• Use IEX as the crude capture stepUse IEX as the crude capture step– Useful when have low level of expression

Anion (Q) or Cation (S)– Anion (Q) or Cation (S)

• Affinity for intermediate– With or without tag removal

• Second IEX or GF for polishing

Variations to the SOP #4Variations to the SOP #4

• Tandem Affinity Purification (TAP‐Tags)Tandem Affinity Purification (TAP Tags)– One affinity tag at N‐terminal, another at C‐terminal

– More commonly used to purify active complexesMore commonly used to purify active complexes

– Can be used for large scale purification

• Especially useful for – Very low expression levelsVery low expression levels

– Getting full length GST fusions [His tag at the C‐terminal]

Experimental DetailsExperimental Details

• Cell lysisCe ys s• Bench‐top affinity column

– Pouringg– Charging with different metals– Loading– Eluting

• SDS‐PAGE of purified protein• Bradford assay for protein concentration• Biochemical assay for activity

Cell Lysis ‐ ExperimentalCell Lysis  Experimental

• Add approximately 10 mls cold MCAC 0 buffer per dd app o ate y 0 s co d C C 0 bu e peg of cell paste– Thoroughly resuspend cells

• Wash out chamber of microfluidizer with water then MCAC 0 buffer

• Pass cells through microfluidizer• Collect cells with minimal extra buffer• Put lysate into centrifuge tubes and balance• Centrifuge at 20,000 rpm for about 30 min

Purification on a Benchtop ColumnPurification on a Benchtop Column• Pour about 5 ml of beads (10 ml of slurry) into a column

• Add ~20 ml water onto top of column and drip through• Charge column with solution of metal (Nickel, zinc or cobalt) salt in watercobalt) salt in water

• Equilibrate into MCAC 0 (about 25 mls)• Load lysate; let it run through.  Collect “Flowthrough”y ; g g• Wash with at least 5 CV (25 ml) of MCAC 40 (40 mMimidazole)

Spot test with Bradford reagent to make sure no more– Spot test with Bradford reagent to make sure no more protein coming off

• Elute protein with step at 250 mM imidazole in MCAC buffer Collect about 2 5 ml fractionsbuffer.  Collect about 2.5 ml fractions– Spot test for protein

AKTAExplorer DemonstrationAKTAExplorer Demonstration

• Lysate will be filtered thru’ a 0.22 micron filterLysate will be filtered thru  a 0.22 micron filter• Column will be washed, charged and equilibrated on the machineequilibrated on the machine

• Sample pump will be used to load column• Column will be washed until OD returns to• Column will be washed until OD returns to around baseline and stabilizes

• Protein will be eluted with a gradient of MCAC• Protein will be eluted with a gradient of MCAC 1000 buffer (1 M imidazole)

• Fractions will be collected throughout run• Fractions will be collected throughout run

SDS PAGESDS PAGE• Load, flowthough, wash and eluted fractions will be applied to a gel for analysiswill be applied to a gel for analysis

• Mix each sample with loading buffer

• Heat to 95°C for about 5 mins

• Spin briefly and load to gelp y g

• Electrophorese at 200 mA for 45 minutes

• Disassemble wash briefly with tap water and• Disassemble, wash briefly with tap water and cover with stain

Bradford AssayBradford Assay

• Make standardsMake standards– Do serial dilutions using positive displacement pipettespipettes

• Dilute protein to approx correct range

Add B df d t d i th hl• Add Bradford reagent and mix thoroughly

• Wait at least 5 mins

• Read OD at 595 nm

Biochemical AssayBiochemical Assay

• Dilute proteinDilute protein

• Mix protein with reagents

i h• Measure in spectrophotometer