ricin purification protocol

8/3/2019 Ricin Purification Protocol

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Purification of ricin



Ricin as a potent toxin

Is an abundant component of Ricinus communis

Highly toxic to mammalian cells

Mechanism of Action

Catalyzes N-glycosidic cleavage of specific adenine residue from 28SrRNA. Thus ribosomes are depurinated and are incapable of proteinsynthesis

Thus, is known as Ribosome Inactivating Protein



Ricin - Heterodimeric type II RIPA chain-32 kDa, called RTAB chain-Galactose binding lectin, 34 kDa, RTB

Ricin uptake

The RTB of ricin binds to both glycoproteins and glycolipids at cellsurfaces that terminate with galactose.

It has two binding sites for galactose and 100·s of ricin molecules maybind per cell.However, a single ricin can inactivate over 1500 ribosomes per minuteand kill a cell.



Purification of Ricin

Ricin was purified from castor bean seeds by affinity chromatographyon guar gum matrix. RCAI is also co purified with Ricin due to galactosebinding ability. The two proteins are separated by GFC on Sephacryl S-300 column

Seeds were soaked in acetic acid for O/N and were crushed to form a

crude homogenate

Homogenate was centrifuged and clear supernatant was removed

Proteins were precipitated by 80% Ammonium Sulfate precipitation



Precipitated proteins were resuspended in PBS and dialyzed to remove

the salt completely

The dialyzate was centrifuged and supernatant was loaded on guar gumcolumn

The unbound proteins were washed by PBS and bound proteins were

eluted with 0.1M lactose.

Fractions containing protein were pooled and dialyzed against PBS to

remove lactose

Sample obtained was concentrated using ultrafilteration and Loaded on tothe Sephacryl S-300 column



Fractions containing ricin were pooled and the amount of the protein wasestimated by Lowry,s Method.

The purified protein was checked for its purity by running a 12.5% SDS-PAgel.

The purified protein obtained was used further for

Iodination reactionEntrapment of ricin in liposomes for cytotoxicity analysis.



0 2 4 6 8 10 12 14 16 18

0.0

0.5

1.0

1.5

2.0

2.5

3.0

A b s o r b

a n c e a t 2 8 0 n m

Fraction number

Elution profile of 80% ammonium sulfate dialysate of castor bean

extract from cross linked guar gum column.



0 20 40 60 80 100 120

0.0

0.5

1.0

1.5

2.0

A b s

o r b a n c e a t 2 8 0 n m

Fraction number

Separation of Ricin and agglutinin using Sephacryl S-300 column



Ricin

Extracted from castor bean

(Ricinus communis)



Ricin is poisonous if inhaled, injected, or ingested, acting as a toxin by the

inhibition of protein synthesis. Ricin is classified as a type 2 ribosome

inactivating protein (RIP). Whereas Type 1 RIPs consist of a single

enzymatic protein chain, Type 2 RIPs, also known as holotoxins, areheterodimeric glycoproteins. Type 2 RIPs consist of an A chain that is

functionally equivalent to a Type 1 RIP, covalently connected by a

single disulfide bond to a B chain that is catalytically inactive, but

serves to mediate entry of the A-B protein complex into the cytosol.

Both Type 1 and Type 2 RIPs are functionally active against ribosomes in

vitro, however only Type 2 RIPs display cytoxicity due to the lectin

properties of the B chain. In order to display its ribosome inactivating

function, the ricin disulfide bond must be reductively cleaved



Ribosome inactivating protein (RIP)

Type 1 RIPs - a single enzymatic protein chain (maize kernel)

Type 2 RIPs (=holotoxins), are heterodimeric glycoproteins. (Ricin) consist of an A

chain that is functionally equivalent to a Type 1 RIP, covalently connected by a S-S

to a B chain serves to mediate entry of the A-B protein complex into the cytosol

Cancer



Day 1-Soak seed in acetic acid

Take shiny seed of castor bean

Decorticate seeds with pestle

weigh- 30 gm

Prepare 5% acetic acid (in distilled water) Soak seed in 10 times volume of

weight of seed at 4 0C for overnight

Prepare Guar gum column- (for 5th day)



Guar gum is a polysacharide made of the sugars galactose and

mannose

1 gm of dry guar gum bind to 20 mg protein

Treatment of Guargum

Take 15 mg Guargum in a beaker

Prepare an emulsion of 4.5 ml Epichlorohydrin 45 ml NaOH in a glass

stoppered reagent bottle and add it to Guargum with vigorous stirring

immediately at 40 0C in water bath

Stirred paste (by spatula) occasionally for 24 hrs



Day 2

Prepare PBS buffer ± will be used from day 3 Morning ± cool mixer jar at 4 0C before use Homogenize in blender at 4 0C for 4 hrs

Its bearing should be moved/rotate by hand- so it will not break when attachedthe mixer. Centrifuge homogenate at 10,400 g (15000 rpm) for 30 mins at 4 0C Scoopu off the upper oily layer Keep 1 ml aliquot at -20 0C Process muslin cloth- (Boil muslin cloth in RO water for 10 mins. Replace the RO water with fresh RO water and further boil for 10 mins.

Dry cloth on filter paper). Filter the supernatant through processed muslin cloth -Note total volume of it Calculate Ammonium sulfate to add for 80% saturation (here 561 gm/lit).

Ammonium sulfate should be added very slowly (2 hrs) on ice bath usingmagnetic stirring. Temperature should be maintained at 4 0C so protein couldnot be denaturated.

Ammonium sulfate is added to ± lower down the heat of solubilization . It prevents denaturation of protein



Day 2-Precipitation of protein

Precool Homogenizer at 4 0C

Homogenize soaked seeds in the same 5% acetic acid (in which it was soaked)in blender at 4 0C till good paste is formed and stirred for 4 hours in magneticstirrer at 4 oC

Process muslin cloth

Centrifuge homogenate at 10,400 g (15000 rpm) for 30 mins at 4 0C

Scoop off the upper oily layer Filter the supernatant through processed muslin cloth

Calculate Ammonium sulfate to add for 80% saturation - add very slowly (2hrs) on ice bath using magnetic stirring at 4 0C

Prepare PBS buffer ± will be used from day 3



For 1 litre of PBS buffer (20 mM)

Solution A-

NaH2PO4 . 2H2O =

NaCl ± 0.15 mM

800 ml of distilled H2O8 g of NaCl

0.2 g of KCl

1.44 g of Na2HPO4 · 12H2O

0.24 g of KH2PO4

Adjust the pH to 7.4 with HCl

Make up volume to 1 litre by distilled H2O



Treatment of Guargum

Take 15 mg Guargum in a beaker

Prepare an emulsion of 4.5 ml Epichlorohydrin 45 ml NaOH and add it

to Guargum with vigorous stirring in capped bottle at 40 C in water bath

Stirred paste for 24 hrsSpread evenly on a filter paper in tray

Keep tray in oven for 6-8 hrs

Break lumps by mortar and pestle

Swell this in deionized water for overnight

Wash extensively (at least 6 times) - yellow to cream color whenepichlorohydrin is removed

Homogenize it for few seconds

Store in 0.02% Na azide at room temperature



Wash column with distilled water

Place glass wool in bottom

Wash it to remove broken particles of glass wool

Fill 50 ml distilled water and mark level Decent water and fill up Guargum up to mark

Allow to settle the gel



Process muslin cloth

Boil muslin cloth in RO water for 10 mins.

Replace the RO water with fresh RO water and further boil for 10 mins.

Dry cloth on filter paper



DAY 3

Cool oakridge in ice

Centrifuge solution at 10,400 g (15000 rpm) for 40 mins at 4 0C

Collect pellete

Suspend pellete in minute volume of PBS (20 mM, pH 7.4)- note down thevolume used of PBS (2 ml).

Keep an aliquot at -20 0C

Proper treatment of dialysis membrane.



Day 3 - Pellete down protein

Cool oakridge in ice

Centrifuge solution at 10,400 g (15000 rpm) for 40 mins at 4 0C

Collect pellete

Suspend pellete in minute volume (2 ml) of PBS (20 mM, pH 7.4)

Dilute this suspension to 24 ml by PBS buffer

Dialyze this in 3.5 liter PBS buffer overnight

Change receptor media at least 3 times



DAY 4

Make 2 ml of this suspension to 24 ml PBS buffer

Dialyze this in 3.5 litre same buffer for 2 days- make at least 3 changes per day(at 10 AM, now 1st change at 1 PM and then 5 PM and 9 PM)

Proper treatment of dialysis membrane after use by azide solution and keep at4 0C.



Day 4 - Dialysis

Change receptor media at least 3 times

Pack Guar gum column



Wash column with distilled water

Place glass wool in bottom

Wash it to remove broken particles of glass wool

Fill 50 ml distilled water and mark level

Decent water and fill up Guargum up to mark

Allow to settle the gel



DAY 5

Check presence of ammonium sulphate by precipitation with BACL2

Centrifuge dialysate at 12000 rpm for 40 mins at 4 0C

Take supernatant (36.5 ml) for protein purification

Keep 1 ml aliquot (0.5 ml) at -20 0C

Guargum column was equilibrated with PBS at a flow rate of 15 ml/hr.

Load supernatant in the column .

After elution- the elute was again loaded in the column on the same flow rate.

Let the solution to elute again and

after elution- Keep column overnight for proper binding of the protein at 40C



Day 5- Centrifuge and load in Guargum column

Equilibrate Guargum column with PBS at a flow rate of 15 ml/hr.

Centrifuge dialysate at 12000 rpm for 40 mins at 4 0C

Take supernatant for protein purification

Load supernatant in the column .

After elution- reloaded elute in the column with same flow rate.

Let the solution to elute again and

Keep column overnight for proper binding of the protein at 4 0C



DAY 6

Wash Guargum column (200 mg protein bind to 1 gm of dry guar gum- whenswelled 1 gm dry guar gum = 7 ml) ± this washing is for elution of non bound proteins ± ricin and lactose will compete for binding ± at higher concentrationof lactose ricin will elute out (rate of washing can be increased upto 20ml/hr)with equilibration buffer (20 mM. PBS) till absorbance at 280 nm become lessthan 0.05

Bound protein was eluted with 0.1 M lactose in PBS.

Collect fractions of 3 ml

Take OD of all fractions at 280 (protein absorb at this wavelength)

Pool the fraction containing ricin and agglutinin and keep at 40C.

Dialyze pooled fractions in 20 mM PBS to remove lactose for 3 change per

day



Day 6 - Wash Guargum column

Wash Guargum column - till absorbance at 280 nm become less than 0.05

Bound protein was eluted with 0.1 M lactose in PBS

Collect fractions of 3 ml

Take OD of all fractions at 280

Pool the fraction containing ricin and agglutinin and keep at 4 0C.

Dialyze pooled fractions (2 day) in 20 mM PBS to remove lactose



DAY 7

Dialysis ± 3 changes per day

Prepare Sephacryl Columns- desalting gel to separate agglutinin and ricin



Sephacryl Columns

Alkyl dextran cross-linked with NN¶-methylene-bis acrylamide



Sephacryl Columns

S-100 HR - separating peptides and small proteins.

S-200 HR and S-300 HR are for purifying antibodies, serum proteins, andmid-size proteins (10-1500Kd).

The tertiary structure of ricin -globular, glycosylated heterodimer of 60-65

kDA. S-400 HR and S-500 HR are used to separate polysaccharides,

macromolecules with extended structures

S-1000 SF - dextrans up to 108 molecular weight, spherical particles up to 400nm, and DNA up to 20 000 base pairs including plasmids, vesicles, andviruses.



Sephacryl Columns

Place glass wool in bottom of column

Wash gel 3 times with PBS (20mM)

Pour in column

Allow to settle



DAY 8

Concentrate protein by ultra filtration.

Equilibrate sephacryl S-300 column wirh 20 mM PBS.



DAY 9

Load (2 ml) concentrated protein in column

ricin purification protocol

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