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 28S rRNA. Thus ribosomes are depurinated and are incapable of protein synthesis Thus, is known as Ribosome Inactivating Protein

Molecular structure of Ricin allows it to bind to mammalian cells, enter via endocytic uptake, and deliver the catalytically active polypeptide into the cell cytosol, where it irreversibly inhibits protein synthesis causing cell death Because of its cytotoxic potency,ricin is being used for selective killing of unwanted cells. Type I RIP- not cytotoxic as they cannot enter eukaryotic cells to reach their ribosomal substrates. Type II RIP- Heterodimeric toxins, interact with cell surface glycosides, enter the cytosol and able to kill the cell by inhibiting protein synthesis.

Ricin - Heterodimeric type II RIP A chain-32 kDa, called RTA B chain-Galactose binding lectin, 34 kDa, RTB

Ricin uptake
The RTB of ricin binds to both glycoproteins and glycolipids at cell surfaces that terminate with galactose. It has two binding sites for galactose and 100s of ricin molecules may bind per cell. However, a single ricin can inactivate over 1500 ribosomes per minute and kill a cell.

Purification of Ricin
Ricin was purified from castor bean seeds by affinity chromatography on guar gum matrix. RCAI is also co purified with Ricin due to galactose binding ability. The two proteins are separated by GFC on Sephacryl S300 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 gum column

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 to the Sephacryl S-300 column

Fractions containing ricin were pooled and the amount of the protein was estimated by Lowry,s Method. The purified protein was checked for its purity by running a 12.5% SDS-PA gel. The purified protein obtained was used further for Iodination reaction Entrapment of ricin in liposomes for cytotoxicity analysis.

Elution profile of 80% ammonium sulfate dialysate of castor bean extract from cross linked guar gum column.

3.0 2.5 Absorbance at 280 nm 2.0 1.5 1.0 0.5 0.0 0 2 4 6 8 10 12 14 16 18

Fraction number

Separation of Ricin and agglutinin using Sephacryl S-300 column

2.0

1.5 Absorbance at 280nm

1.0

0.5

0.0 0 20 40 60 80 100 120

Fraction number

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, are heterodimeric 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 attached the 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 using magnetic stirring. Temperature should be maintained at 4 0C so protein could not 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 magnetic stirrer 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 (2 hrs) 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 ANaH2PO4 . 2H2O = NaCl 0.15 mM

800 ml of distilled H2O 8 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 hrs Spread 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 when epichlorohydrin 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 the volume 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 at 4 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- when swelled 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 concentration of 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 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 (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, and mid-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 400 nm, and DNA up to 20 000 base pairs including plasmids, vesicles, and viruses.

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