EP1456233A1

EP1456233A1 - Haemocyanin from abalone and process of purification thereof

Info

Publication number: EP1456233A1
Application number: EP01942884A
Authority: EP
Inventors: Bhanumathy Manickavasagam
Original assignee: Queensland Bioprocessing Technology Pty Ltd
Current assignee: Queensland Bioprocessing Technology Pty Ltd
Priority date: 2001-06-15
Filing date: 2001-06-15
Publication date: 2004-09-15
Also published as: EP1456233A4; WO2002102844A1

Abstract

An isolated 250 kDa haemocyanin from the black lip abalone (Haliotis ruber). A method of purification of haemocyanin from marine invertebrates using phenyl hydrophobic interaction chromatography (phenyl HIC).

Description

Haemocyanin from Abalone and process of purification thereof.

Technical Field

The present invention is concerned with a novel haemocyanin and, more particularly with a novel haemocyanin isolated from abalone by a novel process. Background Art

Haemocyanin (He) is the blue, copper-containing respiratory protein of many molluscs and arthropods . Haemocyanins are always found freely dissolved in the blood (or hemolymph) . The molluscan haemocyanins have an entirely different structure and arrangement of subunits compared to arthropod haemocyanins. Aerobic metabolism of abalone is supported by gas exchange through gills found in the respiratory cavity. The blood pumped through the gills, via a low-pressure open circulatory system, contains haemocyanin which transports oxygen to respiratory tissues. In open systems blood flows from arteries into the tissue spaces and finally into venous sinuses before being collected in veins and returned to the heart . Oxygenated blood ranges rom pale to strong blue depending on the degree of oxygenation, haemocyanin concentration and species of animal. Dimeric copper pairs in the haemocyanin provide reversible sites for the binding of one oxygen molecule. Haemocyanin is also a source of copper that may lead to inorganic and organic blueing reactions in abalone food processing.

Haemocyanins are arranged into multi-subunit proteins which carry as few as six or as many as several hundred oxygen molecules. Molluscan haemocyanins are extremely large macromolecules having molecular masses of around 4 million daltons (Da) . Molluscan haemocyanins have subunits containing seven or eight oxygen binding functional units. Each globular functional unit is of about 50 kDa and they are arranged like a string of beads. Ten such subunits assemble to form cylindrical decameric whole molecules and in gastropods multiples of two or more decamers may be found. The wall of the decamer has sixty oxygen binding units, and the remaining units form the so- called collar which lies in the centre of the cylinder and, in the case of gastropod haemocyanin, offset to one end. The association of heamocyanin subunits requires divalent cations, either Mg²⁺ or Ca²⁺, as well as competent monomers (Mangum, 1983).

The copper content of molluscan haemocyanins averages around 0.25%, corresponding to 1 gram atom per 25000 daltons of protein. Haemocyanins are potent immunogens which induce the synthesis of large amounts of specific antibodies. The He may exist in associated or dissociated forms (Bartell and Campbell, 1959) . In addition to containing associated or dissociated He molecules, various preparations may contain a number of other immunologically distinct proteins. For instance the hemolymph of the crab may contain at least 5 distinct proteins as well as two electrophoretically distinct He (Horn and Kerr, 1969) .

It has now been found that a novel haemocyanin exists in abalone, which may be isolated by a novel process.

Disclosure of the Invention

According to a first aspect of the present invention there is provided a process for isolating haemocyanin from a marine invertebrate, comprising the steps of :

(1) providing a haemocyanin-containing portion of said marine invertebrate for chromatographic separation; (2) loading a phenyl hydrophobic interaction chro atography (phenyl HIC) column with the haemocyanin- containing portion in order to effect chromatographic separation of haemocyanin from other components of the haemocyanin-containing portion, a haemocyanin fraction being retained on the phenyl HIC column;

(3) eluting the haemocyanin fraction; and

(4) collecting the eluted haemocyanin fraction, wherein the chromatographic separation is effected at a pH less than 8.

Typically, the chromatographic separation is effected with an equilibration buffer with a pH less than 8 and a high ionic strength. The equilibration buffer would usually contain a high concentration of sodium chloride, and would typically be at least 1M for sodium chloride, preferably at least 3M for sodium chloride and sometimes 4M for sodium chloride, or stronger. The equilibration buffer generally also further comprises minor quantities of potassium phosphate, magnesium chloride and calcium chloride.

In a particularly preferred embodiment of the invention the equilibration buffer has a pH of 7, but equilibration buffers with a lower pH may be used. An equilibration buffer with a pH of 6 is used in one embodiment of the present invention, but still lower pHs may be used.

The haemocyanin fraction is retained on the phenyl HIC, whilst other components of the haemocyanin- containing portion flow through the column. Therefore, in a subsequent step the haemocyanin fraction is eluted with an elution buffer which typically has a pH less than 8 and a low ionic strength. The elution buffer may contain minor quantities of potassium phosphate, magnesium chloride and calcium chloride. In a particularly preferred form of the invention the elution buffer has a pH of 7 or 6, but may have a lower pH.

Advantageously, a salt is added to the haemocyanin-containing portion prior to loading it onto the phenyl HIC column. Typically this is done through the addition of sodium chloride, and generally through the addition of a substantial volume of strong sodium chloride solution. Advantageously, a volume half to twice the volume of the haemocyanin-containing portion of sodium chloride is added, and the sodium chloride solution is between 2M and 6M. Minor quantities of magnesium chloride and calcium chloride may be added to the haemocyanin- containing portion.

Advantageously, the eluted haemocyanin fraction is dialysed against deionised water to remove salts . The eluted fraction may also be subjected to ultrafiltration or diafiltration. The product may be freeze-dried or used as a solution.

Advantageously, the marine invertebrate is abalone. Typically the marine invertebrate is selected from the commercial species comprising the black-lip abalone, Haliotis ruber, the brown-lip abalone, Haliotis conicopora, the green-lip abalone, Haliotis laevigita, and Roe's abalone, Haliotis roei .

It is particularly preferred that the haemocyanin portion be blood, typically abalone blood, and preferably the blood of the above named species . In a second aspect of the present invention there is provided a novel haemocyanin when prepared by the process described above.

According to a third aspect of the present invention there is provided a novel haemocyanin isolated from abalone and with a molecular weight of 250 kDa.

Brief Description of the Drawings

Preferred embodiments of the present invention will now be described, by way of example only, with reference to the accompanying Figures, in which: Fig 1 is a chromatogram showing separation of He from East Coast Abalone on Phenyl HIC;

Fig 2 is the absorbance spectrum of East Coast Abalone He column load; Fig 3 is an SDS-PAGE gel of the chromatography fractions from Example 4 and 6 in which the lanes are as follows :

Lane 1 - molecular weight marker

3 - pooled flow through (freeze thawed) 4 - pooled elution (freeze thawed)

5 - flow through fraction l(Day 5 WEC tank storage)

6 - elution fraction 2 (Day 5 WEC tank storage) ;

Fig 4 is a chromatogram showing separation on Phenyl HIC for batch 1 in Example 5;

Fig 5 is a chromatogram showing separation on Phenyl HIC for batch 2 in Example 5;

Fig 6 is an SDS-PAGE gel of the diafiltration retentates of batches 1 and 2 of Example 5 in which the lanes are as follows:

Lane 1 - molecular weight marker

4 - large scale batch 1 retentate

5 - large scale batch 2 retentate

6 - batch 1 (freeze drying trial) 7 - batch 2 (freeze drying trial); and

Fig 7 is a chromatogram showing chromatographic separation of He on Phenyl HIC after freezing and then thawing.

Modes for Performing the Invention

Unless stated otherwise, all experiments were performed at 15°C or on ice.

EXAMPLE 1

CHROMATOGRAPHIC RESIN SELECTION

A number of different resins were trialled for the chromatographic purification of haemocyanin, including the anion exchanger, diethylaminoethyl (DEAE),the cation exchangers carboxymethyl (CM) and sulfoethyl (S) , the size exclusion resin cross-linked sepharose (CL-4B) , and the hydrophobic interaction resins methyl and phenyl.

Blood samples from both freshly shucked animals and abalone blood canned at an abalone food processing plant in Tasmania and air-freighted to Brisbane, Queensland in an insulated container at 4°C were used in the chromatography trials for resin selection. The canned blood was centrifuged at 12000 x g for 10 minutes at 4°C immediately upon arrival, and the pooled supernatant aliquoted into sterile 500 ml containers. A portion was retained at 4°C for purification of He and the remainder stored at -20°C.

During the above chromatography trials, ImM PMSF (phenylmethylsulfonylflouride) and ImM EDTA (ethylenediaminetetraacetic acid) were added to some of the load samples .

Removal of Contaminants Prior to Chromatography The following experiments were conducted to determine whether the removal of contaminants from the abalone blood improved the binding of the He to the different resins tested.

1. The abalone blood sample was ultracentrifuged in a Beckman L8-55 with 50T rotor at

40000 rpm to remove contaminants.

2. He was precipitated by addition of ammonium sulfate to 65% saturation. The ammonium sulfate slurry was collected by centrifugation and redissolved in phosphate buffered saline.

3. The abalone blood sample was ultrafiltered using a Vivascience Vivaflow 50 100 kD ultrafiltration cartridge to half the original volume.

Colour of Abalone Blood

Note was taken of any differences in the colour of the abalone blood obtained at various times. Results

Table 1 - He Purification on Different Resins Under Various Conditions

The yield is defined as the amount of material present in the elution expressedas a percentage of the load.

PB - poor binding

Removal of Contaminants Prior to Chromatography

The ammonium sulfate slurry contained He at a concentration of 22 mg/ml and was dark blue in colour.

There was no loss of He in the ultrafiltration step.

The resin of choice for the chromatography was

Matrex® Cellufine™ Phenyl HIC. The Cellufine™ range of hydrophobic interaction resins is manufactured by Chisso Corporation and marketed under the Matrex®.

Proteins are made up of chains of amino acids, each with a side group attached, some of which are hydrophobic. Proteins order themselves in aqueous solution to achieve minimum free energy by burying most of their hydrophobic groups inside the protein and leaving the charged groups on the outside. Some of the hydrophobic groups will remain exposed on the outside giving localised hydrophobic regions that are available for association with hydrophobic groups on the resin. Hydrophobic interaction chromatography (HIC) is a separation procedure based on the attraction between hydrophobic groups on the protein and a hydrophobic ligand, such as a phenyl group.

Many different ligands are available with a range of hydrophobic properties. The interaction is enhanced with increasing ionic strength of the protein solution and with increasing hydrophobicity of the ligand. Elution of bound protein is effected by decreasing the ionic strength (or salt concentration) of the solution flowing through the column.

Protein Estimation

Protein estimations were carried out using the

Pierce BCA assay. This assay is based on the reduction in alkaline conditions of Cu²⁺ to Cu¹⁺ by protein (biuret reaction) and the colourimetric detection of Cu¹⁺ using bicinchoninic acid (BCA) .

1. An appropriate amount of working reagent was prepared by the mixture of 50 parts of reagent A and 1 part of reagent B. For each sample, 2 ml of working reagent was aliquoted into Johns 5 ml polystyrene tubes.

2. 0.1 ml of each sample (load sample, pooled flow through and pooled elution fractions) was added to a tube and mixed by gentle inversion. The load sample required an initial dilution of 1/5 with elution buffer. A blank was prepared using 0.1 ml elution buffer. The tubes were placed in a preheated water bath at 37°C for 30 minutes then allowed to cool on the bench for 10 minutes.

3. A standard curve was prepared by diluting a stock solution of BSA to a range of concentrations from

25-2000 μg/ml and assaying as described above. . The samples were read on a Biorad Smart Spec 3000 spectrophotometer using the inbuilt BCA protein assay function. This allows the storage of standard curves and automatic calculation of sample concentration.

Disposable UV grade PMMA cuvettes were used for absorbance measurement at 562 nm.

Absorbance Spectrum An absorbance spectrum was scanned from 240 to

460 nm for column load He samples using a Biorad Smart Spec 3000. Samples were initially diluted 1/15 with de- ionised water.

A number of resins were trialled for the commercial purification of He from abalone blood (Table 1) There was no significant difference between the freshly collected and canned blood. The activity of naturally occuring proteases in the haemolymph may lead to degradation of the He aggregates. Therefore the association state of He was assessed by treating the blood with PMSF. EDTA was added as a chelating agent. The presence of one or both of PMSF and EDTA had no significant effect on the protein aggregation as analysed by SDS-PAGE (not shown) .

The removal of contaminants from the abalone blood prior to chromatography did not improve the binding of the He to any of the resins.

It has surprisingly been found that only Phenyl HIC resins are suitable for isolation of He from abalone. There is a need to work at a pH less than 8 for reasons of protein stability, and working at this pH is not conducive to strong binding on the usual resins such as DEAE resin. Likewise, the high sensitivity to pH of the binding to S resin means this resin is not useful, and the high cost and low loading capacity of size exclusion resin means these are not suitable. The hydrophobicity of haemocyanin means that binding is poor to the weakly hydrophobic Methyl HIC resin under normal conditions, and therefore this resin is not suitable.

EXAMPLE 2

ISOLATION AND PURIFICATION OF HAEMOCYANIN FROM EAST COAST

ABALONE (WEC)

Step 1. Abalone Fishing, Storage and Transport

Black-lip abalone (Haliotis ruber) were fished from Storm Bay on the east coast of Tasmania. These animals were shipped to Brisbane, Queensland without tank storage at the process plant in Tasmania. Seven live abalone (batch 1) were air-freighted in March 2001 from Tasmania to Brisbane, Queensland. The abalone were transported from Tasmania to Queensland as a dry consignment. The abalone were placed in sealed, oxygen filled bags with wet foam to keep the humidity high. The animals were held vertically in a head down position by attachment to waxed cardboard sheets. This allows waste products to flow away from the animal. At all times during transport the animals were kept in an insulated container at 4°C.

Upon arrival the abalone will have lost about 15% in body weight due to water loss. If they are returned to tanks promptly, they will regain this weight within 2-3 hours. It is not uncommon for abalone transported dry to arrive alive but to languish once returned to seawater.

Step 2. Live Holding Tank

On arrival, the live animals were transferred to a live holding tank. It measures 1430 mm long X 430 mm wide X 450 mm high, giving a volume of approximately 280 litres . A pump circulates the water through a filter and aeration system while a refrigeration unit controls the water temperature at 10°C.

The tank is sited in a separate room for quarantine purposes and is protected from fluctuations in the external environment . The status and movements of the animals were closely monitored and feeding of seafood pellets was conducted once a week. The abalone have been kept in the live holding tank for over two months with zero mortality. Water filtration is quite efficient and so the tank requires little cleaning.

Abalone can be grown successfully on a very limited budget with this type of setup.

Step 3. Shucking and Method of Blood Collection

The animal is washed under cold running water to remove slime and sand. The animal was turned upside down and shucked by sliding a broad spatula under the foot at the flat region of the shell until the attachment of the foot to the shell was cut. Care was taken not to rupture any internal organs. The spatula was then run gently around the inside edge of the shell to detach the internal organs . The whole animal was then able to be tipped out of the shell.

The guts and other organs (visceral part) were carefully separated from the foot using a scalpel. Care was taken not to rupture any internal organs so as to prevent possible contamination of the blood. The internal organs were further dissected, bagged separately and stored at -20°C for other protein extraction. The mouth area was cut away from the front of the foot with a scalpel, bagged and stored at -20°C.

The foot was rinsed with water and weighed. Several deep incisions were made in the front area of the foot with a scalpel and the foot suspended over a strainer to allow the blood to drain to a collection vessel. Care was taken to avoid bacterial contamination. This was done at 4°C with an initial collection after 1 hour and a further collection after 6 hours.

The foot was either processed immediately or stored at -20°C for the extraction of collagen as described in our co-pending application entitled "Process for

Obtaining Natural Collagen", the contents of which are incorporated herein by reference .

Any remaining organic material was scraped from the inside of the shell, which was rinsed with water and left to dry at room temperature. The shells were stored at room temperature for future work (extraction of proteins) .

The blood was balanced and centrifuged at 12000 x g for 10 minutes at 4°C. The supernatant was decanted, the volume measured, and stored at 4°C in clean, sterile containers. The pellets were discarded. Step 4. Isola tion and Purification of He

Method

A 14 ml Phenyl HIC column was run on a Perseptive Biosystems BioCAD 700E at a flowrate of 1.0 ml/min. The equilibration buffer contained 50mM potassium phosphate, 1M NaCl, ImM MgCl₂, ImM CaCl₂, at pH 6.0. The elution buffer contained 50mM potassium phosphate, ImM MgCl₂/ ImM CaCl₂, at pH 6.0. The cleaning in place solution was 0.5M NaOH.

Sample Preparation

1. The load sample was prepared the day before the chromatography on the BioCAD. 2. 0.5 ml of 4M NaCl was slowly added to 2 ml of centrifuged blood supernatant with constant mixing.

3. 2.5 μl of 1M MgCl₂ + 2.5 μl of 1M CaCl₂ were then added and mixed.

Chroma t ography

1. The resin was equilibrated with 5 column volumes of equilibration buffer.

2. 2 ml of sample (equivalent to 1.6 ml blood) was loaded onto the column. 3. Flow through fractions were collected (4 ml per tube) .

4. The resin was washed with 2 column volumes of equilibration buffer.

5. Step elution was with 100% elution buffer for 4 column volumes.

6. Elution fractions were collected (4 ml per tube) .

7. Cleaning in place was performed with 2 column volumes of 0.5M NaOH . 8. Cleaning in place fractions were collected

(4 ml per tube) . Dialysis

The cleaning in place fractions were pooled and extensively dialysed against de-ionised water to remove traces of sodium hydroxide.

Protein Estimation

Protein concentrations of the chromatography fractions were carried out using absorbance measurements at 340 nm. 1. The absorbance of the load sample, pooled flow through and pooled elution fractions at 340 nm was measured against an elution buffer blank. The load sample required an initial dilution of 1/20 with elution buffer.

2. The haemocyanin concentration is calculated from the extinction coefficient for abalone haemocyanin (ε^1%ι _cm =0.223). A Biorad Smart Spec 3000 spectrophotometer was used with a quartz UV grade cuvette.

Absorbance Spectrum

An absorbance spectrum was scanned from 240 to 460 nm for column load He samples using a Biorad Smart Spec 3000. Samples were initially diluted 1/15 with de- ionised water.

Amino Acid Analysis

Amino acid analysis of east coast He samples (pooled flow through and elution fractions) were done using a Waters amino acid analyser. Samples containing approximately 5 μg of protein were prepared for amino acid analysis.

Results

Table 2 - Appearance of Centrifuged He

Table 3- Yield of He from Phenyl HIC Chromatography (Fig. 1)

Table 4- Amino Acid Analysis of Chromatography Fractions

51 - WEC flow through

52 - WEC elution

The He from the east coast abalone was successfully bound to the resin with a high yield (85%) .

EXAMPLE 3

ISOLATION AND PURIFICATION OF HAEMOCYANIN FROM GREEN-LIP

ABALONE Step 1. Abalone Fishing, Storage and Transport

A single green-lip abalone (Haliotis laevigata) was fished from King Island in Bass Strait and tanked at the process plant for 2 days. The time in the crate (from catch to tank storage) was around 8 hours. The maximum time out of water was 14-15 hours.

The animal was air-freighted in April 2001 from

Tasmania to Brisbane, Queensland as described in Example

2, Step 1.

Step 2. Live Holding Tank

On arrival, the live animal was transferred to a holding tank as described in Example 2 , Step 2.

On the 14^th day the green-lip abalone was able to be slid easily along the floor of the tank and so was removed and shucked.

Step 3. Shucking and Method of Blood Collection This is described in Example 2, Step 3.

Step 4. Isolation and Purification of He

Method

A 5 ml Phenyl HIC column was run on a Biologic LP at a flowrate of 1.5 ml/min. The equilibration buffer contained 50mM potassium phosphate, 3M NaCl, ImM MgCl₂, ImM CaCl₂, at pH 7.0. The elution buffer contained 50mM potassium phosphate, ImM MgCl₂, ImM CaCl₂, at pH 7.0. The cleaning in place solution was 0.5M NaOH.

Sample Preparation

1. The load sample was prepared immediately prior to the chromatography on the Biologic LP.

2. 2 ml of 6M NaCl was slowly added to 2 ml of centrifuged blood supernatant with constant mixing.

3. 4 μl of 1M MgCl₂ + 4 μl of 1M CaCl₂ were then added and mixed. Chromatography

1. The resin was equilibrated with 6 column volumes of equilibration buffer. 2. 2 ml of sample (equivalent to 1.0 ml blood) was loaded onto the column.

3. Flow through fractions were collected (4 ml per tube) .

4. The resin was washed with 3 column volumes of equilibration buffer.

5. Step elution was with 100% elution buffer for 5 column volumes .

6. Elution fractions were collected (4 ml per tube) . 7. Cleaning in place was performed with 2.5 column volumes of 0.5M NaOH .

8. Cleaning in place fractions were collected (4 ml per tube) .

Protein Estimation

Protein concentrations of the chromatography fractions were carried out using absorbance measurements at 340 nm as described in Example 2.

Results

Table 5 - Appearance of Centrifuged He

Table 6- Binding of He to Phenyl HIC Chromatography Resin

The percentage bound is calculated as 100 X (He elution + He CIP) / (He flow through + He elution + He CIP) . This table indicates good binding of green lip abalone haemocyanin to the resin under the conditions tested. The He purification for the green-lip abalone was similar to the east coast animals, with a similar % binding as seen in Table 6.

EXAMPLE 4 ABALONE (EAST COAST) TANK STORAGE TRIAL

Abalone stored in the live holding tank were periodically sacrificed to test the effect of storage on the chromatographic purification of haemocyanin.

Step 1. Abalone Fishing, Storage and Transport This is described in Example 2, Step 1.

Step 2. Live Holding Tank

This is described in Example 2, Step^* 2.

Step 4. Isolation and Purification of He

Method

Sample Preparation

Samples were prepared from animals shucked at the following times:

Day 0 - animal shucked immediately on arrival with no tank storage (control) Day 2 - animal shucked after 2 days of tank storage

Day 5 - animal shucked after 5 days of tank storage.

1. The load sample was prepared immediately prior to the chromatography.

2. An aliquot of centrifuged blood supernatant was taken.

3. An appropriate volume of concentrated NaCl was slowly added with constant mixing to give a final salt concentration of 3M.

4. 1M MgCl₂ and 1M CaCl₂ were then added to give a final concentration of ImM and mixed.

Chroma t ography

1. The resin was equilibrated with 4.5 column volumes of equilibration buffer.

2. 2 ml of sample was loaded onto the column.

3. Flow through fractions were collected (4 ml per tube) .

4. The resin was washed with 3 column volumes of equilibration buffer.

5. Step elution was with 100% elution buffer for 6 column volumes . 6. Elution fractions were collected (4 ml per tube) . 7. Cleaning in place was performed with 2 column volumes of 0.5M NaOH.

8. Cleaning in place fractions were collected (4 ml per tube) .

Protein Estimation

Protein concentrations of the chromatography fractions were carried out using absorbance measurements at 340nm as described in Example 2.

Molecular Weight and Purity

The molecular weight and purity of abalone haemocyanin was evaluated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) . A 4-15% Biorad precast Tris glycine gel was used. SDS-PAGE was performed according to the method of Laemmli (1970) .

The haemocyanin samples (column flow through and elution fractions) were from the Day 5 chromatography. The haemocyanin standard was initially diluted 1/50 in deionised water. All samples were then diluted to half concentration with Gradipore Glycine sample buffer.

The samples were then placed into a boiling water bath for 3 minutes then allowed to cool. The gel was assembled in a Biorad Mini-Protean 3 electrophoresis cell. The inner chamber was filled with SDS glycine running buffer and the samples loaded with an autopipettor and standard yellow tips. The total protein load per well was 2 μg. A molecular weight marker (Biorad broad range prestained marker) was run with each gel. The outer chamber was filled with running buffer to the level of the wells.

The running conditions were 150V constant voltage over 60 minutes with an approximate start current of 50 inA. The gel was then removed from the casing and rinsed with water for around 30 seconds.

The gel was stained with around 50 ml of Gradipore Gradipure stain (based on colloidal G-250 Coomassie blue) overnight with gentle shaking. The gel was destained with frequent changes of water. Bands were generally visible after 5 minutes with about a day required for complete destaining. Permanent storage of gels was achieved by drying between cellophane sheets. The destained gels were soaked in a drying solution of 20% methanol and 2% glycerol with gentle shaking for 15 minutes. Two cellophane sheets per gel were wetted in the drying solution for around 30 seconds. The trimmed gel was clamped between the cellophane sheets in a drying frame and left to stand in an open container at room temperature for 2 days . The gel was then pressed for a number of days prior to display.

Results

Table 7- Effect of Tank Storage of EC Abalone on Purification of He

The percentage bound is calculated as 100 X (He elution + He CIP) / (He flow through + He elution + He CIP) .

The live holding tank was installed to study the effect of the storage of live abalone (east coast) in the tank. The He was analysed from animals shucked after different periods of storage (Table 7) . All samples showed good binding to Phenyl HIC. The SDS-PAGE showed a single band at 250 kDa with 99% purity (Figure 3).

Although the biology of abalone has been widely studied, little is known about the influence of stress on the chemical structure of haemocyanin. Even in a tank rearing system with frequent seawater changes, it is common to observe an accumulation of inorganic nitrogen such as ammonia, nitrite and nitrate (Chen et al, 1989a, Chen et al 1989b) . Ammonia originates from the a monification of organic waste (faeces, excess feed, dead animals) by heterotrophic bacteria that use this waste as a source of nutrients and also from animal excretion resulting from the deamination and transamination of the digested food assimilated by the animals. Abalone excrete ammonia directly into the surrounding water. In an aqueous medium, un-ionised ammonia exists in equilibrium with ionised ammonia and hydroxide ions. This equilibrium depends on pH, temperature and salinity. The un-ionised form is the most toxic because of its readiness to diffuse across cell membranes .

Ammonia may affect gill structure, respiratory function and oxygen consumption in aquatic animals. Oxygen uptake is a critical factor in assessment of stress in fishes. Green-lip abalone were found to be highly sensitive to ammonia as indicated by depressed growth rates and food consumption (Harris et al, 1998) .

For aerobic autotrophic bacteria such as Nitrosomonas sp, ammonia constitutes the metabolic source of energy that is oxidised into nitrite. Nitrobacter sp. oxidises nitrite to nitrate (Sharma and Ahlert, 1977). Nitrite is toxic, particularly in fishing rearing systems, and for molluscs with regard to their haemocyanin (Colt and Armstrong, 1981) .

Abalone appear to be oxygen regulators although starvation is know to limit regulatory ability (Gaty and Wilson, 1986) . Oxygen concentrations can vary in systems subject to high biological oxygen demand (BOD) in which uneaten food and decaying wastes are only removed intermittently, as in some abalone tank systems.

To avoid the above problems the live holding tank used has effective filtration and oxygenation as discussed in one co-pending application entitled "Novel Process", the contents of which are incorporated herein by reference. EXAMPLE 5

VALIDATION OF PURIFICATION OF HAEMOCYANIN BY PHENYL HIC CHROMATOGRAPHY The He purification process was scaled up to a development stage. Two litres of abalone blood was processed using a one litre column of Phenyl HIC resin.

Step 1. Abalone Fishing, Storage and Transport Black-lip abalone (Haliotis ruber) were fished from Storm Bay (April 2001) on the east coast of Tasmania. These animals were shipped directly to the abalone process plant in Tasmania and shucked immediately. A total of 140 abalone (weighing around 90 kg) produced around 4.5 litres of blood. This blood was collected as aseptically as possible in 1000 ml sterile containers.

The blood was air-freighted to Brisbane in an esky and kept at 4°C. Upon arrival, the blood was immediately centrifuged at 12000 X g for 10 minutes at 4°C and the pooled supernatant aliquoted into sterile 500 ml containers. 2.3 litres were retained for purification of He and the remainder stored at -20°C for validation of long-term storage.

Step 2. Isolation and Purification of He Method

A 1000 ml Phenyl HIC column was run at a flowrate of 30 ml/min. The equilibration buffer contained 50mM potassium phosphate, 3M NaCl, ImM MgCl₂, ImM CaCl₂, at pH 7.0. The elution buffer contained 50mM potassium phosphate, ImM MgCl₂, ImM CaCl₂, at pH 7.0. The cleaning in place solution was 0.5M NaOH.

Sample Preparation Two litres of abalone blood was purified in two batches on the Phenyl HIC column. 1. The load sample was prepared immediately prior to the chromatography.

2. 1200 ml of 5M NaCl was slowly added to 1000 ml of centrifuged blood supernatant with constant mixing. 3. 2.2 ml of 1M MgCl₂ + 2.2 ml of 1M CaCl₂ were then added and mixed.

Chroma t ography

1. The resin was equilibrated with at least 5 column volumes of equilibration buffer until a pH between

6.9 and 7.1 is reached.

2. 2200 ml of sample (equivalent to 1000 ml blood) was loaded onto the column.

3. Flow through fractions were collected (400 ml per tube) .

4. The resin was washed with at least 4 column volumes of equilibration buffer until the absorbance of the fractions reached baseline.

5. Step elution was with 100% elution buffer for at least 5 column volumes until the absorbance of the fractions reached baseline.

6. Elution fractions were collected (400 ml per tube) .

7. Cleaning in place was performed with 2 column volumes of 0.5M NaOH.

8. Cleaning in place fractions were collected (400 ml per tube) .

Protein Estimation Protein concentrations of the chromatography fractions were carried out using absorbance measurements at 340nm as described in Example 2.

Molecular Weight and Purity The molecular weight and purity of abalone haemocyanin was evaluated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) . A 4-20% Gradipore iGel precast Tris glycine gel was used. SDS-PAGE was performed according to the method of Laemmli (1970) as described in Example 4.

Ultraf iltration and Dia filtration

A Millipore Prep Scale TFF cartridge was used for the initial concentration and diafiltration steps. A

Vivascience Vivaflow 50 TFF cartridge was used for the final concentration step. The diafiltration buffer contained 83mM sodium phosphate, 150mM NaCl at pH 7.2.

1. The pooled elution was concentrated from 3.6 litres down to approximately 400 ml using the Prep Scale TFF cartridge with a cross-flow rate of 1200 ml/min.

2. This was followed by 5 x volume diafiltration using Prep Scale TFF cartridge. The cartridge was drained and rinsed to retentate.

3. The protein concentrations of retentate and permeate were checked.

4. The retentate was concentrated to approximately 50 mg/ml using the Vivaflow 50 cartridge.

The cartridge was drained and rinsed to retentate.

5. The protein concentrations of retentate and permeate were rechecked.

6. The retentate was sterile filtered through a 0.2 μm filter capsule into sterile container. The protein concentrations of retentate and permeate were checked. The filter was rinsed through with diafiltration buffer to give the required final volume for a He concentration of 30 mg/ml. 7. The He sample was aliquoted into 10 ml sterile vials for dispatch.

Amino Acid Analysis

Amino acid analysis of He samples (batch 1 and 2 diafiltration retentates) was done using a Waters amino acid analyser. Samples containing approximately 5 μg of protein were prepared for amino acid analysis. Results

Table 8- Appearance of Centrifuged Blood

Table 9- Yield of He from Phenyl HIC Chromatography (Figs. 4 and 5)

The percentage yield is calculated as 100 X (He elution) / (He flow through + He elution + He CIP) . This table indicates good binding of He to the resin.

Table 10 - Recovery of He following Ultrafiltration / Diafiltration / 0.2μm Filtration

Fig. 6 is an SDS-PAGE gel of the diafiltration retentates of batches 1 and 2. Table 11- Amino Acid Analysis of Final He Product from Large Scale Process

EXAMPLE 6

FREEZE / THAWING TRIAL OF WEC ABALONE BLOOD

Step 1 Abalone Fishing, Storage and Transport This is described in Example 2, Step 1.

Step 2. Live Holding Tank This is described in Example 2, Step 2.

Step 4. Xsolation and Purification of He

Method

Sample Preparation

An aliquot of the Day 5 centrifuged blood supernatant from Example 4 was frozen at -20°C for a period of 7 days. The sample was thawed and purified.

1. The load sample was prepared immediately prior to the chromatography.

2. An aliquot of centrifuged blood supernatant was taken.

Chroma tography 1. The resin was equilibrated with 8 column volumes of equilibration buffer.

2. 2 ml of sample was loaded onto the column.

3. Flow through fractions were collected (4 ml per tube) . 4. The resin was washed with 4 column volumes of equilibration buffer.

5. Step elution was with 100% elution buffer for 4 column volumes .

6. Elution fractions were collected (4 ml per tube) .

7. Cleaning in place was performed with 3 column volumes of 0.5M NaOH.

8. Cleaning in place fractions were collected (4 ml per tube) . Protein Estimation

Molecular Weight and Purity

The molecular weight and purity of abalone haemocyanin was evaluated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) . A 4-15% Biorad precast Tris glycine gel was used. SDS-PAGE was performed according to the method of Laemmli (1970) as described in Example 4.

Results Table 12- Effect of Freeze / Thawing on

Purification of He

The data from Table 12 (east coast abalone) suggest that freeze/thawing does not adversely affect the chromatographic performance of the haemocyanin in terms of reduced binding and the SDS-PAGE showed a single band at 250 kDa with 99% purity (Fig. 3). This indicates the stability of the protein and the consistency of purification of He. Hence abalone blood can be stored at -20°C with no deleterious effects on He purification.

EXAMPLE 7 FREEZE DRYING TRIAL OF WEC ABALONE BLOOD Method

1. Samples (2 ml) of the diafiltration retentates from batches 1 and 2 were freeze dried for 12 hours. The drying samples were continuously monitored so that they could be removed as soon as drying was complete.

2. The freeze dried samples were resuspended in de-ionised water to their original concentrations and analysed by BCA protein assay (as described in Example 1) and SDS-PAGE (as described in Example 4) .

Results

Table 13 - Appearance of Freeze Dried and Resuspended He

The freeze dried product showed a single band at 250 kDa in gel electrophoresis, and a purity of 99% (Fig. 6) .

Industrial Applicability

The novel haemocyanin of the present invention is useful as a pharmaceutical agent, particularly as an anti-tumour agent, especially for bladder cancer. It is also useful as an ingredient in cosmetic formulations. In addition, it is useful as an immunoadjuvant . It may also be used as a laboratory tool in the life sciences, for example to coat ELISA plates, in chromatography media and in tissue culture media as a replacement for Bovine Serum Albumin. The following references have their disclosure incorporated herein by reference:

References Bartel, A.H. and Campbell D.H (1959) Arch

Biochem. Biophys 82, 232.

Chen J. C, Chen, K.J and Liao J.M. (1989a) Aquaculture 77, 329.

Chen J.C, Liu P.C, Lin Y.T. and Lee C.K (1989b) High-intensive culture study of tiger prawn Peπaeus monodon in Taiwan. In: De Pauw N, Jaspers E. Ackefors H, Wilkins N. (Eds) Aquaculture, a Biotechnology in progress. European Aquaculture Society, Bredene, Belgium, pp 377- 382. Colt J, Armstrong D.A (1981) Nitrogen toxicity to crustaceans, fish and molluscs. In: Allen L.J, Kinney L.J (Eds), proceedings of the Bioengineering Symposium for Fish Culture Section, American Fisheries Society, Bethesda MD pp 34-47. Gaty, G. and Wilson, J.H. (1986) . Aquaculture

56, 229.

Harris, J.O., Maguire, G.B., Edwards, S. and Hindrum, S.M. (1998). Aquaculture 160, 3-4, 259.

Horn B.C and Kerr (1969). Comp.Biochem. Physiol. 29, 493.

Mangum, C.P., (1983). Oxygen Transport in the blood. In: Mantel, L.H. (Ed.); Bliss, D.E. (Series Ed.), The Biology of Crustacea. Vol. 5. Internal Anatomy and Physiological Regulation. Academic Press, New York, pp. 373-429.

Sharma B. and Ahlert C. (1977) Water Res. 11, 879.

Claims

1. A process for isolating haemocyanin from a marine invertebrate, comprising the steps of:

(1) providing a haemocyanin-containing portion of said marine invertebrate for chromatographic separation;

(2) loading a phenyl hydrophobic interaction chromatography (phenyl HIC) column with the haemocyanin- containing portion in order to effect chromatographic separation of haemocyanin from other components of the haemocyanin-containing portion, a haemocyanin fraction being retained on the phenyl HIC column;

(3) eluting the haemocyanin fraction; and

2. A process as claimed in claim 1 wherein the chromatographic separation is effected with an equilibration buffer with a pH less than 8 and a high ionic strength.

3. A process as claimed in claim 2, wherein equilibration buffer has a high concentration of sodium chloride .

4. A process as claimed in claim 3 wherein the equilibration buffer is at least 1M for sodium chloride.

5. A process as claimed in claim 4 wherein the equilibration buffer is 3M for sodium chloride.

6. A process as claimed in claim 5 wherein the equilibration buffer is 4M for sodium chloride.

7. A process as claimed in any one of claims 2 to 6 wherein the equilibration buffer further comprises minor quantities of potassium phosphate, magnesium chloride and calcium chloride.

8. A process as claimed in any one of claims 2 to 7 wherein the equilibration buffer has a pH of 7.

9. A process as claimed in any one of claims 2 to 7 wherein the equilibration buffer has a pH of 6.

10. A process as claimed in any one of claims 1 to 9 wherein the haemocyanin fraction is eluted with an elution buffer which has a pH less than 8 and a low ionic strength.

11. A process as claimed in claim 10 wherein the elution buffer contains minor quantities of potassium phosphate, magnesium chloride and calcium chloride.

12. A process as claimed in either one of claims 10 or 11 wherein the elution buffer has a pH of 7.

13. A process as claimed in either one of claims 10 or 11 wherein the elution buffer has a pH of 6.

14. A process as claimed in any one of claims 1 to 13 wherein a salt is added to the haemocyanin- containing portion prior to loading it onto the phenyl HIC column.

15. A process as claimed in claim 14 wherein sodium chloride is added.

16. A process as claimed in claim 15 wherein a substantial volume of strong sodium chloride solution is added.

17. A process as claimed in claim 16 wherein the volume is a half to twice the volume of haemocyanin- containing portion and the sodium chloride solution is between 2M and 6M.

18. A process as claimed in claim 17 wherein minor quantities of magnesium chloride and calcium chloride are added to the haemocyanin-containing portion.

19. A process as claimed in any one of claims 1 to 18 further comprising the step of dialysing the eluted haemocyanin fraction against deionised water to remove salts .

20. A process as claimed in any one of claims 1 to 19 further comprising the step of subjecting the eluted haemocyanin fraction to ultrafiltration or diafiltration.

21. A process as claimed in any one of claims 1 to 20 further comprising the step of freeze-drying the eluted haemocyanin fraction.

22. A process as claimed in any one of claims 1 to 21 wherein the marine invertebrate is abalone.

23. A process as claimed in claim 22 wherein the marine invertebrate is selected from the group consisting of the black lip abalone, Haliotis ruber, the brown lip abalone Haliotis conicopora, the green lip abalone, Haliotis laevigita, and Roe's abalone, Haliotis roei .

24. A process as claimed in any one of claims 1 to 23 wherein the haemocyanin-containing portion is blood.

25. A process as claimed in claim 24 wherein the haemocyanin-containing portion is abalone blood.

26. A process as claimed in claim 25 wherein the haemocyanin-containing portion is the blood of the black lip abalone, Haliotis ruber, the brown lip abalone Haliotis conicopora, the green lip abalone, Haliotis laevigita, or Roe's abalone, Haliotis roei .

27. A novel haemocyanin when prepared by the process of any one of claims 1 to 26.

28. A novel haemocyanin isolated from abalone, and with a molecular weight of 250kDa.