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WO2006064512A1 - Procédé de fractionnement et de stockage de protéines - Google Patents

Procédé de fractionnement et de stockage de protéines Download PDF

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Publication number
WO2006064512A1
WO2006064512A1 PCT/IN2004/000387 IN2004000387W WO2006064512A1 WO 2006064512 A1 WO2006064512 A1 WO 2006064512A1 IN 2004000387 W IN2004000387 W IN 2004000387W WO 2006064512 A1 WO2006064512 A1 WO 2006064512A1
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WO
WIPO (PCT)
Prior art keywords
proteins
lysozyme
buffer
protein
complex
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IN2004/000387
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English (en)
Inventor
C. S. Ramadoss
Uma S. Maheshwari
Tania Rangel
Kalyan K. Dewan
P. R. Krishnaswamy
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Unichem Laboratories Ltd
Original Assignee
Unichem Laboratories Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Unichem Laboratories Ltd filed Critical Unichem Laboratories Ltd
Priority to PCT/IN2004/000387 priority Critical patent/WO2006064512A1/fr
Publication of WO2006064512A1 publication Critical patent/WO2006064512A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C07K1/32—Extraction; Separation; Purification by precipitation as complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types

Definitions

  • the present invention relates to a process of fractionating and storing proteins from solutioin of peptides (insulin), enzyihes (catalase) ana proteins from crude extracts (E. Co H yeast).
  • proteins like antibodies can also be used to specifically precipitate a particular protein.
  • 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. CoIi 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 js a globular protein containing 129 amino acids, hydro lyses 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- acetylglucosamine (NAG).
  • this invention provides a process of fractionating and storing" proteins from dilute solution of peptides (insulin), enzymes (catalase) and proteins from crude extracts (E. CoIi yeast) comprising: treating a dilute protein solution in buffer- 1 with lysozyme to form a complex between lysozyme and target peptide / protein (insulin/ catalase / protein) at 4-3O 0 C, obtaining said complex by centrifuging, drying the said complex in vacuum and storing at 4-25 0 C, solubilizing said dried materials in buffer-2 , passing the solubilized proteins through either an anion or cation
  • 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 milliliter.
  • the concentration of lysozyme is 0.2-20.0 mg/ml.
  • the anion exchange column is either weak or strong anion exchange matrices.
  • the anion exchange column is a weak anion 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.
  • a process for the concentration/fractionation of peptides (insulin), enzymes (catalase) and proteins from crude extracts comprises addition of hen egg white lysozyme to a splution of protein" in low ionic strength buffer such as 5OmM Tris-HCl pH 8.0.
  • the precipitable complex formed between lysozyme and target peptide/protein is centrifuged at 1000Ox g for 15 min at 4 0 C
  • the complex is then dried under vacuo and stored at 4-25 0 C until further use.
  • the complex is resuspended in Freunds adjuant and injected 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 Sigma 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.
  • the dissociated complex is then passed through a column of anion-exchange resin previously equilibrated with 5OmM 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.
  • the lysozyme-protein complex can be suspended in 5OmM Acetate buffer pH 5.0 containing 0.2M 1 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.
  • 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 (2OmM 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 experiments. The protein content was measured by Bradford method.
  • 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 minimized due to the lesser number of proteins present.
  • the precipitation was higher at lower pH values in the range of 5-6.
  • the yeast Pichia pastoris was grown for 30hr in YPD medium at 30° C. the cells were harvested and washed with 5OmM Tris-Cl pH 7.5. The cells were resuspended in 5OmM 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 5OmM Tris-Cl pH 7.5 and lysozyme in the concentration range of 50 to 500ug were used.
  • 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.
  • 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.
  • the antibody against insulin was raised in rabbits by injecting the insulin- lysozyme complex.
  • 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 5OmM Tris-Cl ⁇ H8.0.
  • the precipitate resuspended in Freunds adjuant and injected into rabbit intramuscularly.
  • the serum was prepared from the blood collected 4 weeks after 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.
  • 1- protein marker 2- E. coli lysate, untreated, 3- E. coli lysate treated with lysozyme- precipitate, 4- E. coli lysate treated with lysozyme- supernantant, 5- S. cerevisiae untreated lysate, 6- S. cerevisiae lysate treated with lysozyme- precipitate , 7- S. cerevisiae lysate treated with lysozyme- supernatant.
  • E.coli cells were grown overnight in 100ml Luria Bertini (LB) medium at 37 0 C. The harvested cells were washed once with 5OmM Tris.HCl (pH 8.0) and resuspended in the same buffer (10ml). The cells were then lysed in a cell disruptor (Constant Sytems) at 10,000 psi. Following lysis the lysate, was centrifuged at 12100 x g at 4 ° C in a Beckmann J2-MC centrifuge for 10 minutes. The supernatant obtained was suitably diluted in 50 mM Tris.HCl (pH8.0) to give an absorbance (A 280 ) of 2.0.
  • a 280 absorbance
  • the treated samples were kept at room temperature for 10 minutes follwed by centrifugation (centrifugation was carried out in an Eppendorf 5415R at 9300 x g, 4 0 C for 10 minutes).
  • the precipitate obtained was resuspended in 0.2 ml of 5OmM Tris.HCl (pH8.0) and samples analyzed by 15% SDS-PAGE.
  • S. cerevisiae cells were grown over-night in 100ml of Yeast extract-peptone- dextrose (YEPD) medium at 3O 0 C.
  • the harvested cells were washed once in 5OmM Tris.Cl pH 7.5 containing ImM EDTA and resuspended in 10ml of the same buffer but also containing 0.5mM DTT and ImM PMSF.
  • the cells were then lysed in a cell disruptor (Constant Sytems) at 10,000 psi. Following lysis the lysate, was centrifuged at 12100 x g at 4 ° C in a Beckmann J2-MC centrifuge for 10 minutes.
  • the supernatant obtained thus was kept for ultra-centrifugation using a Beckman LE-8K, centrifugation was carried out at 4 ° C, 30 minutes, 40,000 rpm using a 7O 1 .1 Ti rotor.
  • the supernatant obtained was suitably diluted in 50 mM Tris.Hcl (pH 7.5) to give an absorbance (A 2 go) of 2.0.
  • the treated samples were kept at room temperature for 10 minutes follwed by centrifugation (centrifugation was carried out in an Eppendorf 5415R at 9300 x g, 4 0 C for 10 minutes).
  • the precipitate obtained was resuspended in 0.2 ml of 5OmM Tris.HCl (pH7.5) and samples analyzed by 15% SDS-PAGE.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

La présente invention décrit une méthode de fractionnement des protéines qui exploite la capacité du lysozyme d'œuf de poule à former un complexe insoluble avec un certain nombre de protéines. En protéomique, le lysozyme peut être employé pour fractionner des extraits non purifiés d’un certain nombre de tissus, dans le but d'obtenir deux populations distinctes de protéines. Le phénomène de précipitation dû à l'insolubilité du complexe peut également être employé afin de concentrer des enzymes en solution diluée et de les stocker sous forme de complexe. Le complexe lysozyme-protéine peut également être employé afin de former des anticorps contre ledit complexe précipité.
PCT/IN2004/000387 2004-12-13 2004-12-13 Procédé de fractionnement et de stockage de protéines Ceased WO2006064512A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/IN2004/000387 WO2006064512A1 (fr) 2004-12-13 2004-12-13 Procédé de fractionnement et de stockage de protéines

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/IN2004/000387 WO2006064512A1 (fr) 2004-12-13 2004-12-13 Procédé de fractionnement et de stockage de protéines

Publications (1)

Publication Number Publication Date
WO2006064512A1 true WO2006064512A1 (fr) 2006-06-22

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IN2004/000387 Ceased WO2006064512A1 (fr) 2004-12-13 2004-12-13 Procédé de fractionnement et de stockage de protéines

Country Status (1)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4104125A (en) * 1977-02-28 1978-08-01 The Green Cross Corporation Process for producing human lysozyme
WO1993024529A1 (fr) * 1992-06-04 1993-12-09 University Of Southern California Facteur neurotrophique derive de l'epithelium retinien pigmente
US6239262B1 (en) * 1998-01-07 2001-05-29 Rensselaer Polytechnic Institute Low molecular weight displacers for protein purification in hydrophobic interaction and reversed phase chromatographic systems
US20020012982A1 (en) * 2000-07-13 2002-01-31 Invitrogen Corporation Methods and compositions for rapid protein and peptide extraction and isolation using a lysis matrix
WO2003023050A2 (fr) * 2001-09-10 2003-03-20 Emd Biosciences, Inc. Procede de recuperation et d'analyse d'une composante cellulaire de cellules cultivees sans besoin de recolter les cellules au prealable

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4104125A (en) * 1977-02-28 1978-08-01 The Green Cross Corporation Process for producing human lysozyme
WO1993024529A1 (fr) * 1992-06-04 1993-12-09 University Of Southern California Facteur neurotrophique derive de l'epithelium retinien pigmente
US6239262B1 (en) * 1998-01-07 2001-05-29 Rensselaer Polytechnic Institute Low molecular weight displacers for protein purification in hydrophobic interaction and reversed phase chromatographic systems
US20020012982A1 (en) * 2000-07-13 2002-01-31 Invitrogen Corporation Methods and compositions for rapid protein and peptide extraction and isolation using a lysis matrix
WO2003023050A2 (fr) * 2001-09-10 2003-03-20 Emd Biosciences, Inc. Procede de recuperation et d'analyse d'une composante cellulaire de cellules cultivees sans besoin de recolter les cellules au prealable

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SMALES C MARK ET AL: "Use of ion-exchange and hydrophobic-interaction chromatography for the rapid purification of lysozyme-estrone glucuronide conjugates", JOURNAL OF CHROMATOGRAPHY B BIOMEDICAL APPLICATIONS, vol. 662, no. 1, 1994, pages 3 - 14, XP009051776 *

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