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WO2016083911A1 - Nouveau procédé de purification de chitinase a par chromatographie d'affinité - Google Patents

Nouveau procédé de purification de chitinase a par chromatographie d'affinité Download PDF

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Publication number
WO2016083911A1
WO2016083911A1 PCT/IB2015/056710 IB2015056710W WO2016083911A1 WO 2016083911 A1 WO2016083911 A1 WO 2016083911A1 IB 2015056710 W IB2015056710 W IB 2015056710W WO 2016083911 A1 WO2016083911 A1 WO 2016083911A1
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Prior art keywords
protein
chitinase
complex
affinity
fragment
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Ceased
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PCT/IB2015/056710
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English (en)
Inventor
Joanna Bereta
Jarosław JUCHA
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Uniwersytet Jagiellonski
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Uniwersytet Jagiellonski
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Publication of WO2016083911A1 publication Critical patent/WO2016083911A1/fr
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Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2442—Chitinase (3.2.1.14)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01014—Chitinase (3.2.1.14)
    • 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
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes

Definitions

  • Chitinases are the enzymes belonging to the conservative glycosyl hydrolase families 18 and 19, These proteins are expressed by multiple organisms, from viruses, via bacteria, plants and insects to mammals. Chitinases are responsible for hydrolysis of the second most abudant natural polysaccharide - chitin, i.e. a linear ⁇ -1,4- linked polymer of N- acetylglucosamine.
  • chitinase purification methods consist in multi-step procedures that include, covering among others, ion-exchange chromatography, molecular exclusion chromatography or affinity chromatography, provided that the recombinant protein has additional fusion tags.
  • Chitinases have been drawing a continuously increasing attention due to their potential use in multiple industrial sectors.
  • the perspective of chitinase application as the environmentally-friendly bioinsecticide would enable replacement of chemical pesticides commonly used in agriculture.
  • This enzyme may be also potentially used for to produce oligosaccharide chitin derivatives.
  • These compounds may be potentially applied in food processing as natural preservatives as well as in pharmaceutical industry as, among others, germicides and fungicides, or as cholesterol reducers (chitosan derivatives). Due to abundance of chitin in natural environment, application of both inexpensive and efficient chitinase purification method, such as the described invention, may allow for generating of new, alternative and renewable energy sources.
  • Chitinase A is an enzyme of exo- and endochitinase activity and demonstrating wide substrate specificity in a broad pH range. This enzyme is produced in insect cells infected with viruses of the family Baculoviridae , which are rod-shape viruses with circular double- stranded DNA genomes. Currently known baculoviruses infect cells of more than 600 invertebrate species and demonstrate species-specific tropism despite high similarity in genome structure and sequence. In particular, the amino acid sequences of baculoviral chitinases display high similarity, which is evidenced by high identity levels between catalytic domains and the presence of an immunoglobulin-like fold.
  • Protein G is a cell wall protein in bacteria of Streptococcus family. It has been proven that this protein binds to the Fc region of the antibodies. To increase affinity to immunoglobulins, multiple modifications of this protein have been introduced and the modified protein G is nowadays commonly used for purification of antibodies, in particular of human and mouse origin, via the affinity chromatography method.
  • the aim of the invention is providing the purification method for chitinase or protein containing a chitinase amino acid sequence.
  • the subject-matter of the invention is a protein complex containing protein G and non-covalently bound chitinase A or a protein containing chitinase-derived amino acid sequence with affinity to protein G.
  • chitinase A should be understood as a protein with 1,4-beta-poly-N-acetylglucosaminidase activity able to decompose chitodextrrn polymers and chitine and containing a catalytic domain as well as a sequence interacting with protein G.
  • chitinase A may contain the immunoglobulin-Iike fold.
  • chitinase A is, in particular, the enzyme derived from Autographa californica nucleopolyhedrovirus (AcMNPV) with a sequence deposited as GenBank ID: CAD79454.1 or its homologue, in particular the enzyme derived from the Baculoviridae family viruses.
  • AcMNPV Autographa californica nucleopolyhedrovirus
  • the protein complex according to the invention is dissociated in acidic water solution (pH below 5), preferably at pH of app. 2, maintaining high biological activity of chitinase.
  • acidic water solution pH below 5
  • chitinase demonstrates amino acid sequence presented as Sequence 1 or a sequence identical in at least 50%
  • protein G demonstrates amino acid sequence presented as Sequence 3.
  • Identity level should be estimated on the basis of comparative analyses provided by the prior art, e.g.: Sangar V, Blankenberg DJ, Altaian N, Lesk AM. Quantitative sequence-function relationships in proteins based on gene ontology. BMC Bioinformatics 2007, 8:294, doi: 10.1186/1471 -2105- 8-294.
  • protein G is additionally immobilized on a stationary phase.
  • chitinase A or its fragment possessing affinity to protein G forms a part of a fusion protein.
  • Another subject-matter of the invention is a method of separation of chitinase A or a protein containing its fragment possessing affinity to protein G from a water solution. This method has following features:
  • a) protein G contacts with water solution containing chitinase A or a protein that contains chitinase fragment possessing affinity to protein G to obtain a protein complex containing protein G and non-covalently bonded chitinase A or a protein that contains the chitinase fragment possessing affinity to protein G.
  • chitinase A or a protein that contains the chitinase fragment possessing affinity to protein G is dissociated from the obtained protein complex
  • chitinase A or a protein that contains the chitinase fragment possessing affinity to protein G is separated from the solution upon completion of dissociation.
  • the protein complex is obtained according to the invention specified above.
  • dissociation in the stage c) is performed in solution of pH 2.0.
  • the separation is carried out using the affinity chromatography method, in witch the chromatographic stationary phase contains immobilized protein G. and preferably in the stage d) a fusion protein that contains the chitinase fragment possessing affinity to protein G is separated.
  • the method according to the invention may be used for a chromatographic purification of different proteins, especially recombinant proteins in a form of fusion proteins consisting of a protein of interest and the amino acid sequence derived from chitinase A possessing affinity to protein G., using the chromatographic method on the column with stationary phase containing immobilized protein G
  • the presented purification method using bacterial protein G is characterised by high specificity, efficiency and simplicity.
  • Fig. 1 presents electrophoretic separation of fractions upon purification of growth media from above insect cells infected with baculovirus. Numbers 1 - 4 denote the number of fraction containing the eluted protein. Final fraction volume was 1 ml. 25 ⁇ of each fraction was loaded onto the gel. Proteins were visualized by staining with Coomassie Brilliant Blue.
  • Fig. 2 presents the result of protein analysis, using the Western blotting method, obtained upon exposure of chemiluminescent signal onto x-ray film.
  • Numbers 1 - 4 detnote the number of fraction containing the eluted protein.
  • Final fraction volume was 1 ml, 25 ⁇ of each fraction was loaded onto the gel. Proteins were visualized by labeling with anti-Fc antibodies conjugated to horseradish peroxidase, followed by detection using chemiluminescent substrate for horseradish peroxidase.
  • Fig. 3 presents identification of reducing sugars released upon digestion of chitosan with chitinase A (Fehling's reaction).
  • a - chitosan subjected to enzymatic reaction with chitinase A yellow - greenish pellet color due to reduction of Cu ions to Cu ions.
  • B - chitosan not subjected to chitinase A activity blue pellet color due to non-reduced Cu 2+ ions).
  • the chart legend presents different concentrations of used chitinase (500 pM - 7.8 pM) used with constant substrate quantity (7.5 ⁇ ).
  • the chart legend presents chitinases derived from different organisms analyzed with a view to capacity to form a complex with protein G.
  • Example 1 Chitinase A purification, that includes obtaining of a complex with protein G.
  • insect cells SF9 or High FiveTM were cultivated as a suspension culture in ESF 921 medium from ExpressionSystems until density of 2 million of cells/ml of medium was obtained;
  • the cells were infected with baculovirus (MOI-2), following by 120-hour culture at 27°C with mixing (140 rpm).
  • MOI-2 baculovirus
  • the chromatographic column containing immobilized protein G (Protein G Sepharose 4 Fast Flow from GE Healthcare Life Sciences) was equilibrated with 20 mM phosphate buffer, pH 7.1 (made of Bishop reagents) in quantity of 5 times the column volume; e) the column was loaded with previously prepared protein formulation at flow rate of 1 ml/min at 4°C;
  • g) protein was eluted by water solution of 0.1 M citric acid, pH 2.0 (Bioshop).
  • Protein G is a protein of app. 21.6 kDa molecular mass, whereas the molecular mass of chitanase A is approximately equal to 65 kDa. Molecular mass of the produced complex is estimated for app. 87 kDa.
  • Fig. 1 presents separation of the collected fractions in the polyacrylamide gel SDS-PAGE (reducing conditions) stained with Coomassie Briliant Blue.
  • Example 2 Identification of chitinase A purified using a method that includes obtaining of a complex with protein G.
  • Protein fractions separated in polyacrylamide gel as show in Example 1 were subjected to electrotransfer onto a PVDF membrane, which was then blocked with 5% skim milk solution in TBS buffer.
  • the membrane was incubated with anti-mouse IgG antibody solution (Fc region-specific), conjugated with horseradish peroxidase, for 3 hours in ambient temperature. Following washing, the membrane was covered with a chemiluminescent substrate for horseradish peroxidase and the signal was exposed onto the x-ray film.
  • Fig. 2 presents the obtained result.
  • the obtained sequence was compared to sequences of proteins collected in the UniProtKB/Swiss-Prot database. The analysis demonstrated clearly that the obtained protein is Autographa californica nucleopolyhedrovirus (AcMNPV)-derived chitinase A.
  • AcMNPV Autographa californica nucleopolyhedrovirus
  • Example 3 Analysis of enzymatic activity of chitinase A purified with method that includes obtaining of a complex with protein G.
  • chitinase purified using the affinity chromatography with the use of protein G is biologically active.
  • the first test was carried out by determination of reducing sugars produced upon chitosan hydrolysis in effect of enzymatic activity of the purified chitinase (Fehling's reaction). In this test, Cu 2+ ions are reduced (bluish color) to Cu + ions (yellow-greenish color) due to the presence of reducing sugars in the analyzed sample (Fig. 3).
  • the second test was measurement of fluorescence intensity of a product (4* methylumbelliferone) produced upon enzymatic digestion of a substrate being the chitine analogue (4-meu ⁇ ylumbelliferone-p-D-N i N ⁇ N''-t-riacetylchitotrioside, MUF-triNAG), by the purified chitinase.
  • the chart (Fig. 4) presents the dependence of fluorescence intensity (proportional to product quantity) as a function of the reaction time. Constant substrate concentration (7.5 ⁇ ) and variable amounts of chitinase (from 500 pM to 7.8 pM) were used in the assay. Fluorescence was induced at 360 nm, whereas its measurement was carried out at 455 nm for 100 minutes.
  • chitinase derived from Autogtapha californica nucleopolyhedrovirus (enzyme purified as specified in example 1), Streptomyces griseus (Sigma- Aldrich), Trichoderma viride (Sigma- Aldrich)-derived chm'nases and recombinant human chitinase (Sino Biological Inc.) to form a complex with protein G.
  • a direct ELISA test was performed. The wells of the ELISA test plate were coated with chitinases derived from different organisms by using protein solutions of 8 ⁇ g/ml concentration in 50 mM borate buffer of pH 8.3; coating was performed overnight at 4°C.
  • amino acids at positions 14-121 form immunoglobulin-like fold, absent in the sequence of the remaining tested chitinases.
  • the bioinforrnatic analysis carried out demonstrates that at least several dozens of chitinases produced by different organisms has an amino acid fragment demonstrating homology of at least 50% to the immunoglobulin-like fold of AcMNPV chitinase A.
  • chitinase A which belongs to the group of enzymes applied in the industry for, among others, plant protection and potentially also as fungicidal therapeutic and enzymatic tool in many other domains (including, among others, productions of medicines and fuels).
  • the invention enables purification of the enzyme produced using any expression system.
  • chitinase derived from the baculovirus protein expression system commonly used for recombinant proteins production, is purifiable.
  • the fragment of chitinase A amino acid sequence is possible to be used as the new type of a fusion tag suitable for purification of recombinant proteins using protein G.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

Cette invention concerne un complexe protéique contenant une protéine G et qui est lié de manière non covalente à une chitinase A ou une protéine contenant un fragment de chitinase ayant de l'affinité pour la protéine G et le procédé de séparation de la chitinase A ou d'une protéine contenant un fragment de chitinase ayant de l'affinité pour la protéine G à partir d'une solution aqueuse.
PCT/IB2015/056710 2014-11-28 2015-09-03 Nouveau procédé de purification de chitinase a par chromatographie d'affinité Ceased WO2016083911A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PLP.410286 2014-11-28
PL410286A PL235436B1 (pl) 2014-11-28 2014-11-28 Kompleks białkowy zawierający białko G i chitynazę A, sposób wyodrębniania z roztworu wodnego chitynazy A oraz jego zastosowania

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3862007A (en) * 1973-03-15 1975-01-21 Canadian Patents Dev Extraction of chitinase
WO1997047752A1 (fr) 1996-06-14 1997-12-18 Icos Corporation Materiaux et procedes de production de produits de chitinase
US6352850B1 (en) 1999-10-27 2002-03-05 Agency Of Industrial Science & Technology Chitinase and method for preparing the same

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3862007A (en) * 1973-03-15 1975-01-21 Canadian Patents Dev Extraction of chitinase
WO1997047752A1 (fr) 1996-06-14 1997-12-18 Icos Corporation Materiaux et procedes de production de produits de chitinase
US6352850B1 (en) 1999-10-27 2002-03-05 Agency Of Industrial Science & Technology Chitinase and method for preparing the same

Non-Patent Citations (21)

* Cited by examiner, † Cited by third party
Title
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MOB T; ABRAHMSÉN L,; NILSSON B: "Staphylococcal protein A consists of five IgG-binding domains", EUR J BIOCHEM., vol. 156, no. 3, 2 May 1986 (1986-05-02), pages 637 - 43
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R E. HAWTIN; T. ZARKOWSKA; K, ARNOLD; C. J. THOMAS; G. W. GOODAY; L. A. KING; J. A. KUZIO; R. D. POSSEE: "Liquefaction of Autographa californica nucleopolyhedrovirus-infected insects is dependent on the integrity of virus-encoded chitinase and cathepsin genes,", VIROLOGY, vol. 238, no. 2, November 1997 (1997-11-01), pages 243 - 253
R RAO; L. FIANDRA; B. GIARDANA; M DE EGUILEOR,; T. CONGIU; N. BURLINI; S. ARCIELLO; G. CORRADO; F. PENNACCHIO: "AcMNPV ChiA protein disrupts the peritrophic membrane and alters midgut physiology of Bombyx mori larvae", INSECT BIOCHEM. MOL. BIOL., vol. 34, no. 11, November 2004 (2004-11-01), pages 1205 - 1213
R. E. HAWTIN; K ARNOLD; M. D. AYRES; P. M. DE A. ZONOTTO; S. C. HOWARD,; G, W. GOODAY; L. H. CHAPPELL; P.L A. KITTS; L. A. KING; R: "Identification and Preliminary Characterization of a Chitinase Gene in the Autographa californica Nuclear Polyhedrosis Virus Genome", VIROLOGY, vol. 212, no. 2, October 1995 (1995-10-01), pages 673 - 685
SANGAR V; BLANKENBERG DJ; ALTMAN N; LESK AM: "Quantitative sequence-function relationships in proteins based on gene ontology", BMC BIOINFORMATICS, vol. 8, 2007, pages 294
YURIY MIHAYLOV STOYKOV ET AL: "Chitinase biotechnology: Production, purification, and application", ENGINEERING IN LIFE SCIENCES, vol. 15, no. 1, 3 December 2014 (2014-12-03), DE, pages 30 - 38, XP055232680, ISSN: 1618-0240, DOI: 10.1002/elsc.201400173 *

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