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WO2011014051A1 - Procédé de contrôle de la dégradation protéolytique de protéines recombinées - Google Patents

Procédé de contrôle de la dégradation protéolytique de protéines recombinées Download PDF

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Publication number
WO2011014051A1
WO2011014051A1 PCT/MY2009/000105 MY2009000105W WO2011014051A1 WO 2011014051 A1 WO2011014051 A1 WO 2011014051A1 MY 2009000105 W MY2009000105 W MY 2009000105W WO 2011014051 A1 WO2011014051 A1 WO 2011014051A1
Authority
WO
WIPO (PCT)
Prior art keywords
protease
protein
niv
recombinant protein
recombinant
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/MY2009/000105
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English (en)
Inventor
Beng Ti Tey
Fui Chin Chong
Wen Siang Tan
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.)
Universiti Putra Malaysia (UPM)
Original Assignee
Universiti Putra Malaysia (UPM)
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 Universiti Putra Malaysia (UPM) filed Critical Universiti Putra Malaysia (UPM)
Priority to PCT/MY2009/000105 priority Critical patent/WO2011014051A1/fr
Priority to MYUI2010006296A priority patent/MY178484A/en
Publication of WO2011014051A1 publication Critical patent/WO2011014051A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6818—Sequencing of polypeptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/115—Paramyxoviridae, e.g. parainfluenza virus

Definitions

  • the present invention relates to a method for controlling proteolytic degradation of recombinant nucleocapsid protein (NCp) of Nipah virus (NiV).
  • protease inhibitor cocktails and specific protease inhibitors are important tools for microbial proteomic studies.
  • protease inhibitors interact with their target proteases by binding to its active site or modify an amino acid residue of the protease active site, resulting in the formation of a stable inactive protease inhibitor complex.
  • the NCp of NiV has been successfully produced in microbial system, and it is highly antigenic and immunogenic [see Ong et al. (2002), Biochem MoI Biol Biophys 6:347-50].
  • Recombinant protein of the NCp gene has potential to be developed as a diagnosis tool for NiV infection and to replace the high risk crude formalin-inactivated NiV antigen.
  • the major problem during the production and purification of the NCp of NiV is the low recovery yield due to the proteolytic degradation.
  • a targeted strategy to inhibit the endogenous host proteolytic degradation recombinant NCp of NiV is presented in this invention.
  • the present invention is directed to a method to identify the specific endogenous proteases that cause the proteolytic degradation of the target recombinant protein (NCp) by adding the specific protease inhibitors to the buffer system.
  • the proteolytic degradation activity was inhibited and as a result substantial improvement in the recovery of the recombinant NCp of NiV.
  • the method of the present invention enables substantial improvement in recovery yield and requires short process time to optimise the formulation of buffer system which is able to inhibit the proteolytic activity.
  • the present invention is directed to a method to identify the specific endogenous protease that causes the proteolytic degradation of a recombinant protein and inhibit its activity; the method comprises the steps of: (a) identifying the size of a degraded protein with an immunodetection method; (b) analysing amino acid sequence of the recombinant protein to identify the specific proteases that cause the degradation of the recombinant protein; (c) confirming the specific endogenous protease identified with immunodetection method; and (d) inhibiting proteolytic degradation of the recombinant protein with a specific protease with a specific protease inhibitor.
  • the inhibitor is selected from the group consisting of: phenylmethylsulphonyl fluoride (PMSF), 4-(2-aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF) and aprotinin.
  • PMSF phenylmethylsulphonyl fluoride
  • AEBSF 4-(2-aminoethyl) benzene sulfonyl fluoride hydrochloride
  • aprotinin phenylmethylsulphonyl fluoride
  • the inhibitor is protease inhibitor
  • concentration of phenylmethylsulphonyl fluoride comprises from 0.8 niM to 1.2 mM.
  • Figure 1 illustrates an example of the SDS-PAGE gel (A) and Western blot (B) of the
  • NCp of NiV precipitated with ammonium sulphate NCp of NiV precipitated with ammonium sulphate.
  • Figure 2 illustrates an example of predicted serine cleavage site (underlined) of the
  • NCp gene of NiV SEQ.ID No.l
  • Figure 3 illustrates an example of the SDS-PAGE gel (A) and Western blot (B) for detecting the presence of potential protease in E.coli BL21(DE3).
  • Figure 4 illustrates an example of the result of the effect of addition of protease inhibitors on the yield of NCp of NiV using Western blot analysis.
  • Figure 5 illustrates an example result of the dose dependence of the PMSF supplementation on the recovery yield of the NCp of NiV from E.coli lysate.
  • NCp of NiV refers to the nucleocapsid protein of Nipah Virus.
  • NCp polypeptide encompasses any polypeptide derived from the full length and naturally occurring NiV-NCp polypeptide listed herein as SEQ ID NO:1.
  • the NiV-NCp polypeptide exemplified in this application is provided as example and do not limit the intent of this invention to include other polypeptide.
  • inhibitors means reducing, slowing or interfering with a process.
  • abbreviation “CIpP” refers to the caseinolytic peptidase.
  • polypeptides are useful in the present (a sequence listing according to ST.25 is also included in the application of the present invention):
  • SEQ ID NO: 1 amino acid sequence of full length mature protein of NCp of NiV i k. ⁇ >., ( i ⁇ u 'l ' ntuit i'i > ii in ⁇ c'Hi »i Ic
  • the inventors have utilized a bioinformatics tool to identify the proteolytic cleavage site and potential endogenous protease by analysing the amino acids sequence that deduced from the NCp gene of NiV, obtained from Gene Bank (Accession number: AJ564621).
  • the potential endogenous protease that caused the cleavage of target peptide and its cleavage site from the amino acid sequence of NCp of NiV was successfully deduced.
  • the prediction of the right protease that attacks the target protein and the supplementation of the specific inhibitor to the buffer system has substantially inhibited the activity of the endogenous proteases and improved the recovery yield of the recombinant NCp of NiV. This method improved the recovery yield substantially and it requires short process time to optimise the formulation of buffer system which is able to inhibit the proteolytic activity.
  • Figure 1 illustrates that two major protein bands were detected in the E. coli lysate by the swine anti-NiV serum: the expected 63 kDa foil length NCp polypeptide and a much smaller 45 kDa NCp protein. This Western blot results confirm that this smaller protein band is the degraded form of the NCp polypeptide.
  • NCp constitutes 70% of the total NCp expressed in E. coli BL21(DE3).
  • SDS-PAGE gel and B Western blot of the NCp of NiV precipitated with ammonium sulfate. Lane 1, molecular weight markers in kDa; Lane 2, NCp. Swine anti-NiV sera were used to detect the presence of recombinant NCp in E. coli cell lysate.
  • Figure 2 illustrates the predicted serine cleavage site (underlined) of the NCp gene of NiV by the bio informatics program.
  • the protease cleavage sites of the NCp of NiV (accession number AJ564621) of SEQ ID NO: 1 are shown with the following symbols.
  • * chymotrypsin (C-term to Phe/Tyr/Trp/Met/Leu, not before Pro)
  • . chymotrypsin (C-term to Phe/Tyr/Trp, not before Pro)
  • o proteinase K.
  • Figure 3 illustrates the presence of the CIpP, a serine proteases, in the cell lysate.
  • A SDS-PAGE gel and
  • B Western blot for detecting the presence of potential proteases in E. coli BL21 (DE3). Lane 1, molecular weight markers in kDa; Lane 2, E. coli cell lysate.
  • the Western blot (B) shows that the CIpP is indeed presence in the cell lysate and most probably responsible to the degradation of the N protein.
  • FIG. 4 illustrates the effects of the addition of protease inhibitors on the yield of NCp using Western blot analysis.
  • PMSF is the most effective inhibitor among all the serine protease inhibitors (PMSF, AEBSF and aprotinin).
  • the recovery yield after adding PMSF was about 2-fold higher than the control (sample without any protease inhibitor supplementary).
  • the yield of NCp treated with AEBSF and aprotinin was about 1.3 times higher than the control.
  • the lysate sample was treated with each of the indicated protease inhibitors at the following concentrations: (1) none, (2) 1 mM
  • E. coli strain BL21(DE3) harbouring plasmid pTrcHis 2 expressing the NCp was grown overnight in 1 L of Luria Bertani (LB) broth containing ampicillin (50 ⁇ g/ml) at 25°C, pH 7 with shaking.
  • LB Luria Bertani
  • IPTG isopropylthio- ⁇ -D- galactoside
  • the cells were harvested by centrifugation at 8000 x g (Avanti JLA-16.250, Beckman-Coulter, USA) for 20 min. Prior to the cell disruption, the cell pellet was washed with distilled water and centrifuged again at 8000 x g (Avanti JLA- 16.250, Beckman-Coulter, USA) for 20 min at 4 0 C.
  • Example 3 Example 3:
  • E. coli cell pellet was resuspended in extraction buffer (25 mM HEPES buffer pH 8) supplemented with 0.2 ⁇ g/ml lysozyme and 4 mM MgCl 2 .
  • the initial crude cell homogenate sample was added immediately with specific protease inhibitors as listed in Table 1 that inhibit the potential protease and then subjected to sonication for 30 s with 60 s intervals in an ice bath for a duration of 50 min.
  • the sonication was operated using an ultrasonic homogenizer (Labsonic U-Ultra 50, B. Braun, Germany) at 20 kHz equipped with a needle titanium probe (40 T) of 4 mm diameter and 127 mm length (Model 853 811/5).
  • the cell lysate was clarified by centrifugation at 18,000 x g.
  • the recombinant protein was precipitated with ammonium sulfate at 30% saturation.
  • dialysis was carried out with Tris-NaCl buffer (50 mM Tris, 100 mM NaCl; pH 8.0).
  • the dialysed protein solution was then assayed for total protein and NCp concentration.
  • Table 1 Preparation of the protease inhibitors stock
  • Example 4 The SDS-polyacrylamide gel (SDS-PAGE) analysis was performed under denaturing condition using a Mini-Protean 3 apparatus (Bio-Rad, USA).
  • the reducing agents used in the electrophoresis buffer was 0.1% (w/v) SDS.
  • Acrylamide of 12% (w/v) were used for resolving and stacking gels, respectively.
  • the gels were stained with Coomassie ® Brilliant Blue (CBB) R-250 (ICI Ltd.) and destained with destaining solution containing 10% (v/v) methanol and 10% (v/v) acetic acid until a clear background was obtained.
  • CBB Coomassie ® Brilliant Blue
  • BSA Bovine-serum albumin
  • C Quantitation of NCp of NiV was determined by Western blots method Proteins were transferred to nitrocellulose membranes by using the Transblot SD ® semidry transfer cell (Bio-Rad, USA), as described above. The NCp concentrations from various samples were estimated by comparing the intensity of the bands from Western blots with the help of an internal standard of the NCp purified using sucrose gradient ultracentrifugation.
  • a volume is the intensity data inside a defined boundary drawn on the images. The intensity of the data inside the boundary and that of other objects can be compared using the Volume Analysis report.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un procédé permettant d’améliorer la récupération d’une protéine N recombinée du virus Nipah produite dans Escherichia coli, selon lequel l’identification et l’inhibition des protéases spécifiques dans le lysat cellulaire sont obtenues par une utilisation intégrée d’un outil bio-informatique et d’un dispositif expérimental d’inhibition des protéases.
PCT/MY2009/000105 2009-07-30 2009-07-30 Procédé de contrôle de la dégradation protéolytique de protéines recombinées Ceased WO2011014051A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/MY2009/000105 WO2011014051A1 (fr) 2009-07-30 2009-07-30 Procédé de contrôle de la dégradation protéolytique de protéines recombinées
MYUI2010006296A MY178484A (en) 2009-07-30 2009-07-30 A method for controling proteolytic degradation of recombinant proteins

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Application Number Priority Date Filing Date Title
PCT/MY2009/000105 WO2011014051A1 (fr) 2009-07-30 2009-07-30 Procédé de contrôle de la dégradation protéolytique de protéines recombinées

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011056060A3 (fr) * 2009-11-06 2011-12-15 Universiti Putra Malaysia Protéine de matrice recombinée du virus nipah

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006091610A2 (fr) * 2005-02-23 2006-08-31 The Brigham And Women's Hospital, Inc. Inhibiteurs de l'infectiosite des virus enveloppes
WO2007017293A2 (fr) * 2005-08-11 2007-02-15 Digilab Biovision Gmbh Procedes de recherche systematique de proteases et de leurs substrats

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006091610A2 (fr) * 2005-02-23 2006-08-31 The Brigham And Women's Hospital, Inc. Inhibiteurs de l'infectiosite des virus enveloppes
WO2007017293A2 (fr) * 2005-08-11 2007-02-15 Digilab Biovision Gmbh Procedes de recherche systematique de proteases et de leurs substrats

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DIEDERICH S ET AL: "Role of endocytosis and cathepsin-mediated activation in Nipah virus entry", VIROLOGY, ACADEMIC PRESS,ORLANDO, US, vol. 375, no. 2, 5 June 2008 (2008-06-05), pages 391 - 400, XP022665850, ISSN: 0042-6822, [retrieved on 20080314] *
DIEDERICH SANDRA ET AL: "The nipah virus fusion protein is cleaved within the endosomal compartment", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOCHEMICAL BIOLOGISTS, BIRMINGHAM, US, vol. 280, no. 33, 1 August 2005 (2005-08-01), pages 29899 - 29903, XP009126687, ISSN: 0021-9258 *
PAGER C T ET AL: "A mature and fusogenic form of the Nipah virus fusion protein requires proteolytic processing by cathepsin L", VIROLOGY, ACADEMIC PRESS,ORLANDO, US, vol. 346, no. 2, 15 March 2006 (2006-03-15), pages 251 - 257, XP024896723, ISSN: 0042-6822, [retrieved on 20060315] *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011056060A3 (fr) * 2009-11-06 2011-12-15 Universiti Putra Malaysia Protéine de matrice recombinée du virus nipah

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