Form 2
THE PATENTS ACT, 1970
COMPLETE SPECIFICATION
[See Section 10]
'^PROCESS OF FRACTIONATING AND STORING PROTEINS'
Vnichem Laboratories Ltd., an Indian company of Mahalaxmi Chambers, 22 Bhulabhai Desai Road, Mumbai - 400026, Maharashtra, India
The following specification particularly describes and ascertains the nature of this invention and the manner in which it is to be performed.


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The present invention relates to a process of fractionating and storing proteins from dilute solution of peptides (insulin), enzymes (catalase) and proteins from crude extracts (E.coli yeast).
BACKGROUND
The isolation of a protein from any sources involves several fractionation steps in which a protein precipitating agent would be often used. The most commonly used agent being ammonium sulphate. In this precipitation process the protein of interest gets partially purified & also gets concentrated. The use of protamine sulphate instead of ammonium sulphate is not uncommon. Sometimes the precipitation of protein is achieved by isoelectric point precipitation or by addition of solvents as well. The problem with these treatments is that not all proteins are stable to these types of treatments.
In addition to practices of using small molecules, some proteins, like antibodies can also be used to specifically precipitate a particular protein. Similarly some of the glycoproteins can be separated from a mixture of proteins using specific lectins. These however have limited scope of applicability.
The object of this invention is to provide a single precipitating agent lysozyme for precipitating protein from crude extract of E.coli yeast, plant and mammalian cell extracts, restriction enzymes, catalase and soyabean lipoxygenase. Furthermore the ability of lysozyme to selectively precipitate proteins present in certain extracts could be utilized in the study of proteomics.
Lysozyme is a globular protein containing 129 amino acids, hydrolyses peptidoglycan found in the cell walls of some bacteria. In the peptidoglycan, the enzyme cleaves the bond between N- acetyl muramic acid (NAM) and.N-
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acetylglucosamine (NAG). While this enzyme is found in all bodily secretions and plants, egg white is the most abundant source. It is presumed to play a protective role against bacterial infection. The enzyme is routinely used in the plasmid isolation technique. The enzyme has been investigated extensively from the structural point of view
To achieve the said objective this invention provides a process of fractionating and storing proteins from dilute solution of peptides (insulin), enzymes (catalase) and proteins from crude extracts (E.coli yeast) comprising:
treating a dilute protein solution in buffer-1, as herein described,
with lysozyme to form a complex between lysozyme and target
peptide / protein (insulin/ catalase / protein) at 4-3 0°C,
obtaining said complex by centrifuging,
drying the said complex in vacuum and storing at 4-25°C,
solubilizing said dried materials in buffer-2, as herein described,
passing the solubilized proteins through either an anion or cation
exchange column to recover lysozymes,
eluting the bound proteins using buffer - 3, as herein described,
and
separating the proteins on 2-D polyacrylamide gel electrophorasis.
The buffer-1 is 50 mM tris-HCl, pH in the range 5-9.
The dilute solution of protein is in concentration range of 0.1-10.0 milligrams protein per mililiter.
The concentration of lysozymes is 0.2-20.0 Mg/ml.
The anion exchange column is either weak or strong anion exchange matrices.
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The anion exchange column is a weak cation exchanger like DEAE-Cellulose.
Buffer-2 is a low ionic strength buffer, 50 mM tris-HCl buffer, ph ranges between 7-8 containing 0.2 M NaCl/
Buffer-3 is higher ionic strength buffer, 50 mM tris-HCl buffer, pH ranges between 7-8 containing 1-1.5 M NaCl.
The solution of protein is obtained from the extraction of various tissues.
Detailed description
A process for the concentration/fractionation of peptides (insulin), enzymes (catalase) and proteins from crude extracts (e.g. E.coli, Yeast etc.) comprises addition of hen egg white lysozyme to a solution of protein in low ionic strength buffer such as 50mM Tris-HCl pH 8.0.
The precipitable complex formed between lysozyme and target peptide/protein (insulin/catalase/proteins) is centrifuged at lOOOOx g for 15 min at 4°C
The complex is then dried under vacuo and stored at 4-25°C until further use.
The complex is resuspended in Freunds adjuant andinjeeted into laboratory animals like rabbits to raise the antibody for the peptide antigen (3insulin/catalase) precipitated by lysozyme. Freund's Adjuvant is a commercially available product produced by Sigtna-Aldrich Fine Chemicals, USA and is used to enhance immune response of the administered antigen.
The lysozyme- insulin complex or the lysozyme-catalase is then treated with buffer of sufficient ionic strength to dissociate the complex.
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The dissociated complex is then passed through a column of anion-exchange resin previously equilibrated with 50mM Tris-HCl buffer pH8.0. The column is charged with the solution containing dissociated lysozyme-insulin or lysozyme-catalase complex. The unbound lysozyme is recovered for reuse. The bound insulin or catalase is recovered from the column by increasing the ionic strength of the eluting 50 mM Tris-HCl buffer pH8 0 by including sodium chloride in the buffer up to a concentration of 1.5 M.
Alternatively, the lysozyme-protein complex can be suspended in 50mM Acetate buffer pH 5.0 containing 0.2M NaCl and the suspended material is centrifuged. The soluble fraction containing the lysozyme is passed through a cation exchange resin to recover the lysozyme. The insoluble precipitate contains all the proteins that formed complex with lysozyme.
The protein thus obtained is analyzed on a polyacrylamide gel electrophoresis. If a mixture of protein is precipitated as in the case of E.coli extract or yeast extract the proteins in the complex is analyzed by 2D electrophoresis or profiled on a suitable FPLC/HPLC column with an appropriate solvent system.
it
The invention will now be described with reference to the following examples
Example-1
Precipitation of proteins from E.coli extract by lysozyme: The E.coli strain BL21* was grown overnight in 100ml of LB medium at 37° C The cells were harvested and resuspended in lysis buffer (20mM TrisCl pH 8.0 containing ImM EDTA & ImM PMSF) in one-tenth volume of the culture broth and lysed in a Dyna mill using a pressure of 20 psi. The lysate was spun at 10000 rpm at 4° C for 10 min. the supernatant was used in the precipitation
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experiments. The protein content was measured by Bradford method. For the actual experiment, a series of 7 independent incubations consisting of 20mM Tris-Cl pH 8.0, 500ug of E.coli protein and lysozyme ranging from 50 to lOOOug were used. The mixture was kept at room temperature (25° C) for lOrnin and the turbidity monitored at 600 rim. The turbidity increased with increasing amount of lysozyme in the reaction mixture (Fig. 1) The reaction rnixture was then centrifuged and the precipitate formed was analyzed on SDS-polyacrylamide gel electrophoresis. It can be seen from the gel picture (Fig.2) that a number of proteins are precipitated as the concentration of the lysozyme is increased up to about 500ug beyond which no further effect is observed. Thus two populations are obtained one precipitable by lysozyme & the other that apparently has no interaction with lysozyme.
This can facilitate the identification of more number of proteins present in the extract by two-dimensional electrophoresis as interference from other proteins can be rninimized due to the lesser number of proteins present.
Furthermore, it can be readily seen from the polyacrylamide gel electrophoretic analysis that some of the proteins get concentrated and thus making their identification easier.
The ability of lysozyme to precipitate certain proteins can be used to concentrate proteins from dilute solutions.
The interesting thing about this precipitation phenomenon is that both larger proteins (Mr >100000) and smaller proteins (Mr < 10000) are precipitated by lysozyme.
The precipitation was higher at lower pH values in the range of 5-6.
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Example-2
Precipitation of proteins from Yeast Extract
The yeast Pichia pastoris was grown for 30hr in YPD medium at 30° C. the cells were harvested and washed with 50mM Tris-Cl pH 7.5. The cells were resuspended in 50mM Tris-Cl pH 7.5 containing ImM EDTA, ImM PMSF & 0.5mM DTT. The cells were lysed under 20psi. The lysate was centrifuged 45000rpm at 4° C. The supernant was used for precipitation by lysozyme. For precipitation experiments, yeast extract (0.5mg/ml) in 50mM Tris-Cl pH 7.5 and lysozyme in the concentration range of 50 to 500ug were used. The turbidity increased with increasing concentration of lysozyme. The samples were centrifuged & the precipitate and the supernant from each of the incubation were analyzed by electrophoresis. Maximum precipitate was observed with 500ug of lysozyme. Experiments with S. cerevisiae extract also showed similar precipitation pattern. The over all about 50-60% of the proteins were precipitated from both yeast extract.
Example-3
I^cipitation of catalase;
In this experiment precipitation of purified catalase, an enzyme that catalysis the degradation of hydrogen peroxide is demonstrated. Here again, 500ug of catalase was taken in 20mM Tris-Cl pH 8.0 and to it varying concentrations of lysozyme ranging from 20 to 50Qug.was added.
The turbidity after lysozyme addition increased with increasing concentration of lysozyme. After the removal of the complex the activity remained in the
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supernant decreased. At a lysozyme concentration of 500ug there was only 10-15% catalase activity detectable in the supernant.
The stoichiometry for the complex formation between catalase & lysozyme was calculated to be 1:16. Since the enzyme has 4 subunits, it appears that each subunit binds at least 4 lysozyme molecules.
The precipitate formed has been resolubilised in 0.2M sodium phosphate buffer pH 8.0 and assay for catalase activity. The activity was fully recovered after the resolubilisation suggesting the intactness of the precipitated enzyme. In fact the complex could be stored for at least a week at room temperature after lyophilisation and after reconstitution the enzyme activity was recovered.
ExampIe-4
Precipitation of insulin:
The commercially available insulin after desalting was used. To 500ug of insulin increasing amount of lysozyme was added. As in other examples cited above, the samples appeared turbid soon after the addition of lysozyme. Maximum precipitation was observed with lOOOug of lysozyme. The stoichiometry of this precipitation reaction is 1:1.
ExampIe-5 Antibody production
The antibody against insulin was raised in rabbits by injecting the insulin-lysozyme complex. For this purpose 0.5 mg of insulin was precipitated with lmg of lysozyme. The complex formed is removed by centrifugation at the 10000 rpm for 15 min. The pellet was washed with 50mM Tris-Cl pH8.0. The precipitate resuspended in Freunds adjuant and injected into rabbit intramuscularly. The serum was prepared from the blood collected 4 weeks after
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the injection. The serum was used to detect the insulin by immunobloting. The detection of insulin in the blot showed that the antibody to insulin has been successfully raised using the insulin-lysozyme complex.

We claim:
1. A process of fractionating and storing proteins from dilute solution of
peptides (insulin), enzymes (catalase) and proteins from crude extracts
{E.coli yeast) comprising:
treating a dilute protein solution in buffer-1, as herein described,
with lysozyme to form a complex between lysozyme and target
peptide / protein (insulin/ catalase / protein) at 4-3 0°C,
obtaining said complex by centrifuging,
drying the said complex in vacuum and storing at 4-25°C,
solubilizing said dried materials in buffer-2, as herein described,
passing the solubilized proteins through either an anion or cation
exchange column to recover lysozymes,
eluting the bound proteins using buffer - 3, as herein described,
and
separating thejjgoteins on 2-D polyacrylamide gel electrophorasis.
2. A process as claimed in claim 1 wherein buffer-1 is 50 mM tris-HCl, pH in the range 5-9.
3. A process as claimed in claim 1 wherein the dilute solution of protein is in concentration range of 0.1-10.0 milligrams protein per mililiter.
4. A process as claimed in claim 1 wherein the concentration of lysozymes is 0.2-20.0 Mg/ml.
5. A process as claimed in claim 1 wherein the anion exchange column is either weak or strong anion exchange matrices.
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6. A process as claimed in claim 1 wherein anion exchange column is a weak cation exchanger like DEAE-Cellulose.
7. A process as claimed in claim 1 wherein buffer-2 is a low ionic strength buffer, 50 mM tris-HCl buffer, ph ranges between 7-8 containing 0.2 M NaCl.
8. A process as claimed in claim 1 wherein buffer-3 is higher ionic strength buffer, 50 mM tris-HCl buffer, pH ranges between 7-8 containing 1-1.5M NaCl.
9. A process as claimed in claim 1 wherein solution of protein is obtained from the extraction of various tissues.
10 A process of fractionating and storing proteins from dilute solution of peptides (insulin), enzymes (catalase) and proteins from crude extracts (E.coli yeast) substantially as herein described with reference to the foregoing examples 1 to 4.
Dated this 29th day of October, 2003

Shanti Kumar
of ANAND & ANAND, Advocates
Agents for the Applicants