JP5288902B2 - Cell surface display method using chitin binding domain - Google Patents

Description

  The present invention relates to a method for presenting molecules on the surface layer of a cell.

  A target protein such as an enzyme and an anchor protein serving as a scaffold for immobilization can be genetically fused and expressed, whereby the protein can be presented on the cell surface of an organism such as yeast.

  Microorganisms presenting various proteins including enzymes on the cell surface have been created (for example, Patent Documents 1 to 21 for yeast, Patent Document 22 for Escherichia coli, Corynebacterium genus bacteria, for example) Patent Document 23 and Patent Document 24) regarding lactic acid bacteria. When the microorganism is yeast, the GPI anchor adhesion recognition signal sequence of the cell surface localized protein or the sugar chain binding protein domain of the cell surface localized protein can be used (Patent Documents 1 to 21). In the case of bacteria (for example, Escherichia coli, Corynebacterium, lactic acid bacteria, etc.), part of the outer membrane localized protein can be used. For example, PgsA, a poly-γ-glutamate biosynthetic enzyme, is a protein localized in the cell surface layer, and can be presented on the surface layer by fusing the target protein to the C-terminal side (Patent Documents 22 to 24).

  By displaying the target protein on the surface of the yeast cell, the protein can be handled easily. The microorganism displaying the enzyme on the surface layer can be used as an immobilized enzyme agent because the enzyme is immobilized on the surface layer simply by culturing. And it can collect | recover and reuse after reaction.

Neisseria gonorrhoeae (for example, Aspergillus oryzae) are useful microorganisms that have the highest protein production ability among microorganisms and are known as GRAS (Generally Recognized As Safe). Due to such protein-producing ability, development of useful protein production methods by Aspergillus has been actively conducted. For example, Patent Document 25 describes a method for efficiently secreting and producing a protein. For the production and use of useful proteins, application of the useful surface layer display technology as described above is also desired for Neisseria gonorrhoeae.
JP-A-11-290078 JP 2002-17368 A Japanese Patent Application Laid-Open No. 2002-176979 JP 2002-253267 A JP 2003-235579 A JP 2004-49014 A JP 2004-194559 A JP 2004-305096 A JP 2004-305097 A JP 2005-58010 A JP 2005-176605 A JP 2005-245334 A JP 2005-245335 A JP 2005-31426 A JP 2006-136223 A JP 2006-174767 A JP 2006-262724 A JP 2007-20539 A JP 2007-89506 A JP 2007-300194 A International Patent Application Publication No. 02/085935 JP 2005-31426 A JP 2007-89506 A JP 2006-262724 A Japanese Patent Application Laid-Open No. 2007-174963

  An object of this invention is to provide the surface layer presentation technique which can be utilized suitably also in Aspergillus oryzae like Aspergillus oryzae.

The present invention provides a method of presenting a protein on the cell surface of a microorganism, the method comprising:
Introducing into the microorganism an expression unit DNA comprising a secretion signal, a chitin binding domain, and a DNA each encoding the protein to be presented; and culturing the microorganism into which the expression unit DNA has been introduced, A step of expressing on the cell surface of the microorganism.

  The present invention also provides a recombinant vector for displaying a protein on the cell surface of a microorganism, the recombinant vector comprising a secretion signal, a chitin-binding domain, and a DNA encoding the protein to be displayed. Containing expression unit DNA.

  In one embodiment, the protein is fused to the N-terminal side of the chitin binding domain.

  In another embodiment, the protein is fused to the C-terminal side of the chitin binding domain.

  In yet another embodiment, the protein to be presented is fused to both the N-terminal side and the C-terminal side of the chitin-binding domain.

  In one embodiment, the chitin binding domain is a peptide region comprising the amino acid sequence set forth in SEQ ID NO: 1.

  The present invention further provides a microorganism having a protein displayed on the surface of a cell produced by the above method or obtained by introducing the above recombinant vector.

  In one embodiment, the microorganism is Aspergillus oryzae.

  ADVANTAGE OF THE INVENTION According to this invention, the surface layer presentation technique which can be utilized suitably also in Aspergillus oryzae like Aspergillus oryzae is provided.

  In the present invention, a chitin binding domain (also referred to herein as “CBD”) can be used as an anchor protein. An anchor protein is a protein that has a role of binding to the cell wall of a cell and orienting the cell from the surface layer to the outside of the cell. A “chitin binding domain (CBD)” refers to any domain that can bind to chitin present on the cell surface.

  A chitin binding domain is a peptide region containing the amino acid sequence of sequence number 1, for example. The amino acid sequence described in SEQ ID NO: 1 is a sequence derived from a chitin-binding domain in the region from amino acid positions 481 to 562 of Saccharomyces cerevisie chitinase (CTS1; DDBJ accession number U17243-9). However, the origin of the use in the present invention does not matter. The above “peptide region containing the amino acid sequence described in SEQ ID NO: 1” means that the amino acid sequence described in SEQ ID NO: 1 is further added, deleted, and / or substituted unless the chitin binding ability is lost. Including those that are. The number of residues to be added, deleted, substituted, etc. can be one or more (eg, any from 1 to 10 residues).

  A target protein to be displayed on the cell surface (hereinafter also simply referred to as “target protein”) is fused to the chitin-binding domain. The protein of interest may be fused to either the N-terminal side or the C-terminal side of the chitin binding domain. The same or different types of proteins of interest may be fused to both the N-terminal side and the C-terminal side of the chitin binding domain. The target protein may be directly bound to the chitin binding domain, or may be bound via a linker.

  The type of protein intended to be presented on the cell surface is not limited. Here, the target protein includes any molecule composed of amino acids regardless of the molecular weight, and does not exclude peptides composed of fewer amino acids. A protein that is originally not localized on the cell surface of the microorganism but is arranged for the purpose of fixing to the cell surface of the microorganism is preferred. For example, enzymes (eg lipases, amylases (eg glucoamylase, α-amylase, β-amylase etc.), cellulases (eg cellulase, hemicellulase etc.)), antibodies, ligands, fluorescent proteins, protein A and And derivatives thereof.

  The target protein may be determined to be fused to the N-terminal side or the C-terminal side of the chitin-binding domain depending on its characteristics. For example, a protein having an active center on the C-terminal side, such as lipase, is preferably fused to the C-terminal side of the chitin binding domain.

  A secretion signal is further linked to the N-terminal side of the fusion between the chitin-binding domain and the protein of interest as described above. Here, the secretory signal generally refers to an amino acid region containing a lot of amino acids rich in hydrophobicity, which is bound to the N-terminus of a protein (secretory protein) secreted extracellularly (including periplasm). The secreted protein is removed when it is secreted from the cell through the cell membrane to the outside of the cell. As the gene encoding the secretion signal, any secretion signal that works effectively in the host cell in which the protein is expressed can be used. If the protein of interest is inherently secreted, a secretion signal derived from that protein can be used. For example, a secretion signal derived from a glucoamylase gene (glaA) having a high secretion ability is preferably used. When the host cell is Neisseria gonorrhoeae (for example, Aspergillus oryzae), for example, the secretion signal (tglA) derived from triacylglycerol lipase of Aspergillus oryzae is preferably used. A signal sequence of α-agglutinin or a-agglutinin of yeast (eg, Saccharomyces cerevisiae) can also be used.

  A region of up to 28 amino acids counted from the C-terminal side of a pro sequence consisting of 97 amino acid sequences of Rhizopus oryzae-derived lipase (ROL) as described in Patent Document 25 (in the present specification, “ N28 region ") may also be used to aid extracellular secretion. The N28 region can be located between the secretory signal sequence and the fusion of the chitin binding domain and the protein of interest. The N28 region can be suitably used particularly in filamentous fungi (for example, Aspergillus oryzae).

  In order to express a protein of interest in a host cell, a DNA encoding an expression unit comprising a chitin binding domain, a protein of interest, and a secretion signal, and optionally an N28 region (herein referred to as “expression unit DNA”). Is also introduced into cells.

  Expression unit DNA can be prepared by synthesizing and binding DNAs encoding the chitin-binding domain, the protein of interest, and the secretion signal, and if necessary, the N28 region, by techniques that can be commonly used by those skilled in the art. In order to prepare the expression unit DNA, a primer is prepared based on the sequence information described in this specification and the known sequence information (secretion signal and nucleotide sequence encoding the protein of interest), and chromosomal DNA or cDNA The desired fragment can be obtained by PCR using as a template. The binding can be performed using an appropriate restriction enzyme, a linker or the like.

  The introduction of DNA means that DNA is introduced into a cell and expressed. Methods for introducing DNA include transformation, transduction, transfection, cotransfection, electroporation and the like. The introduced DNA may be in the form of a plasmid, or may be inserted into a chromosome by being inserted into a host gene or undergoing homologous recombination with a host gene.

  Construction of a recombinant vector suitable for a host can be performed according to techniques commonly used in the fields of molecular biology, biotechnology, and genetic engineering. As the plasmid for the recombinant vector, any plasmid suitable for the host can be used.

  The host for surface layer display expression is not particularly limited as long as it is a cell having a cell wall, such as a bacterium, a fungus, or a plant cell. A microorganism rich in chitin in the cell surface layer (or cell wall) is preferred. Preferred microorganisms rich in such chitin include, but are not limited to, Neisseria gonorrhoeae (for example, Aspergillus oryzae) and yeast (for example, Saccharomyces cerevisiae).

  In the recombinant vector, a promoter corresponding to a unit that controls transcription / translation can be incorporated upstream of the expression unit DNA 5 ′ side, more preferably a terminator 3 ′ side downstream. As the promoter and terminator, any promoter and terminator known to function in a microorganism used as a host is used. When the host cell is a filamentous fungus (eg, Aspergillus oryzae), this promoter is preferably derived from a highly expressed filamentous fungal gene. A number of preferred high-expressing fungal genes are provided, for example, by the following examples: amylase, glucoamylase, alcohol dehydrogenase, xylanase, glyceraldehyde-phosphate dehydrogenase or cello from Aspergilli or Trichoderma Biohydrase gene. The most preferred high expression genes for these purposes are Aspergillus niger glucoamylase gene, Aspergillus oryzae TAKA-amylase gene, Aspergillus nidulans gpdA gene or Trichoderma -The cellobiohydrase gene of Trichoderma reesei. When the host cell is Aspergillus oryzae, preferably P-glaA142 or P-No8142 (these are the glaA (Hata, Y. et al., Gene, 1992, 108, pp.145-150) gene and No. 8AN (Ozeki, K. et al., Biosci. Biotechnol. Biochem., 1996, Vol. 60, pp. 383-389) gene) is used. Examples of the terminator include a terminator derived from a gene encoding α-glucosidase, a terminator derived from a glucoamylase gene (glaA or glaB), and the like. Regarding yeast, for example, a promoter and terminator of glyceraldehyde 3'-phosphate dehydrogenase (GAPDH), a promoter and terminator of phosphoglycerate kinase (PGK), and the like can be mentioned. Examples of such starting material plasmids include GAPDH (glyceraldehyde 3'-phosphate dehydrogenase) promoter sequence and GAPDH terminator sequence plasmid pYGA2270 or pYE22m, or UPR-ICL (isocitrate lyase upstream region) sequence and Examples include plasmid pWI3 containing Term-ICL (terminator region of isocitrate lyase) sequence, plasmid pGK404 containing PGK promoter and terminator sequence, and the like.

  The recombinant vector can further include a selectable marker gene. The selectable marker gene can be selected from a large number of marker genes useful for transformation of, for example, filamentous fungi (eg, Aspergillus oryzae). These markers include, for example, the amnS (acetamide) gene, auxotrophic marker genes (eg argB, trpC or pyrG), and antibiotic resistance genes (eg providing resistance to bleomycin, hygromycin B or G418) However, it is not limited to these. Preferable marker genes are marker genes derived from Aspergillus filamentous fungi, and include niaD, argB, sC, ptrA, pyrG, amdS, aureobasidin resistance gene, benomyl resistance gene, hygromycin resistance gene and the like. NiaD is particularly preferable from the viewpoint of the insertion efficiency of the target gene into the host chromosomal DNA. As for yeast, a shuttle vector with E. coli is preferable from the viewpoint of simplification of DNA acquisition. As a starting material for producing a vector, for example, it has a replication origin (Ori) of a yeast 2 μm plasmid and a replication origin of ColE1, and a yeast selection marker (for example, a drug resistance gene, an auxotrophic marker gene ( For example, it is more preferable to have HIS3, LEU2, URA3, LYS2, etc.) and E. coli selectable markers (drug resistance genes, etc.).

  Transformation of the host can be performed by a method commonly used by those skilled in the art. The procedure for producing a transformant can be performed according to techniques commonly used in the fields of molecular biology, biotechnology, and genetic engineering. In the case of filamentous fungi (eg gonococci such as Aspergillus oryzae), it can preferably be transformed using the protoplast-polyethylene glycol (PEG) method, a transformation protocol developed for Aspergillus. Examples of yeast include a method using lithium acetate and a protoplast method.

  The transformant can be cultured by appropriately selecting a medium and culture conditions suitable for growth of the transformant or production of a desired protein according to a method commonly used by those skilled in the art.

  The surface layer presentation technology using the chitin binding domain as described above is a conventional surface layer presentation technology (for example, use of a GPI anchor attachment recognition signal sequence of a cell surface localized protein, a sugar chain binding protein domain of a cell surface localized protein). Use etc.).

  Microorganisms presenting proteins on the cell surface can be used for the purpose of bringing the protein expressed on the cell surface into contact with a substrate and binding the substrate or converting the substrate into another substance. For example, when the protein is an enzyme, it may be provided as an enzyme agent containing cells that express the enzyme. As such an enzyme agent, a cell containing the enzyme on the cell surface and a suspension containing a medium capable of maintaining the cell, and a cell on which the enzyme is presented on the cell surface are cryopreserved or freeze-dried, and further dried at a low temperature. Can be mentioned.

  In addition, a combinatorial library in which various proteins with randomly introduced mutations are presented on the surface layer of different cells can be prepared. From such a library, it is also possible to quickly and easily select a clone having the optimum properties according to the purpose. The combinatorial library here refers to a collection of various mutants in which mutations are randomly introduced at the gene level and the diversity of proteins is artificially expanded.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, this invention is not limited by this Example.

(Example 1: Preparation of EGFP-CBD fusion expression strain)
PCR using the genomic DNA of Saccharomyces cerevisiae W303-1B strain (obtained from American Type Culture Collection (ATCC)) as a template and the primer pairs shown in SEQ ID NO: 2 (forward) and SEQ ID NO: 3 (reverse) Was performed to amplify the chitin binding domain (CBD).

  On the other hand, a pIS1-ss-N28-EGFP vector, which is a plasmid containing genes encoding the secretion signal (tglA) derived from triacylglycerol lipase of Aspergillus oryzae, the N28 region, and the modified green fluorescent protein (EGFP), Prepared (obtained from Laurel Wreath Corporation).

  CBD obtained by PCR amplification was cleaved with restriction enzymes NotI and SwaI, ligated to pIS1-ss-N28-EGFP vector similarly cleaved with restriction enzymes NotI and SwaI, and plasmid pIS1-sN-EGFP-L-CBD was Obtained. This plasmid contains a gene encoding CBD linked via a linker to the C-terminal side of EGFP, which is an expression target protein.

Using the protoplast-PEG method, plasmid pIS1-sN-EGFP-L-CBD was introduced into Aspergillus oryzae IF4 niaD ^- strain (obtained from Laurel Wreath Co., Ltd.). The obtained transformant was cultured for about 7 days in CD-NO ₃ medium in which NaNO ₂ in Czapek-Dox medium (CD medium) was replaced with NaNO ₃ . Subsequently, 100 ml of dextrin-peptone-yeast extract liquid medium (DPY liquid medium) was inoculated and cultured with shaking at 30 ° C. and 150 rpm for 10 days.

(Comparative Example 1: Preparation of EGFP secretion expression strain)
A transformant was prepared in the same manner as described in Example 1, except that the pIS1-ss-N28-EGFP vector was used for transformation of Aspergillus oryzae IF4 niaD ⁻ strain. By this transformation, an EGFP secretion expression strain was obtained.

(Example 2: Confirmation of expression of EGFP-CBD fusion protein by Western blotting)
First, cells from the respective culture solutions of the EGFP-CBD fusion expression strain of Example 1 and the EGFP secretion expression strain of Comparative Example 1 were disrupted by sonication, and centrifuged at 14000 rpm at 4 ° C. for 5 minutes. A culture supernatant fraction, a soluble fraction and a cell wall fraction were obtained. These fractions were subjected to Western blotting using an anti-EGFP antibody as the primary antibody and an anti-mouse IgG antibody as the secondary antibody, and analyzed for expression of the EGFP-CBD fusion protein. As a control, Aspergillus oryzae IF4 niaD ^- was also used untransformed strain.

FIG. 1 shows an electrophoretic photograph of the result. In this electrophoresis photograph, lanes 1 to 3 are culture supernatants, lanes 4 to 6 are soluble fractions, lanes 7 to 9 are cell wall fractions, and lanes 1, 4, and 7 are EGFP-CBD fusion expression strains ( example 1), lanes 2, 5 and 8 are EGFP secretory expression strain (Comparative example 1), lanes 3,6,9 Aspergillus oryzae IF4 niaD ^- represents the results of the untransformed strain. As shown in FIG. 1, in the EGFP secretion expression strain, a band at a position of about 30 kDa corresponding to the molecular weight of EGFP appeared only in lane 2 as the culture supernatant and did not appear in lane 8 as the cell wall fraction. In contrast, in the EGFP-CBD fusion expression strain, a band appeared at a position of about 150 kDa in lane 7, which is a cell wall fraction. This confirmed that the EGFP-CBD fusion expression strain expressed the EGFP-CBD fusion protein on the cell wall.

(Example 3: Confirmation of protein surface display by fluorescence microscope observation)
EGFP secreted expression strain of EGFP-CBD fusion expression strains and Comparative Example 1 Example 1, and Aspergillus oryzae IF4 niaD was used as a control ^- the culture of untransformed strains, at 30 ° C. with 1 × PBS solution 24 After time treatment, it was observed under a fluorescence microscope. By comparison with observation under a differential interference microscope, it was found that EGFP-derived green fluorescence was localized on the cell surface in the EGFP-CBD fusion expression strain. The EGFP secretion expression strain and the untransformed strain did not show such fluorescence distribution. Thereby, in the EGFP-CBD fusion expression strain, it was shown that EGFP-CBD was presented on the cell surface layer.

  Furthermore, after the cells were treated with proteinase K at 30 ° C. for 24 hours, the cells were again observed under a fluorescence microscope. FIG. 2 shows fluorescence micrographs of proteinase K-treated and untreated EGFP-CBD fusion expression strain and EGFP secretion expression strain. The left column of FIG. 2 shows the EGFP secretion expression strain, the right column shows the EGFP-CBD fusion expression strain, the upper row shows the untreated strain (1 × PBS solution treatment), and the lower row shows the proteinase K treatment strain. In the EGFP secretion expression strain, the change in fluorescence due to the proteinase K treatment was not so much seen, whereas in the EGFP-CBD fusion expression strain, the EGFP-derived fluorescence was present on the surface of the untreated strain, After proteinase K treatment, the existing fluorescence disappeared. Thereby, in the EGFP-CBD fusion expression strain, it was further confirmed that EGFP-CBD was presented on the cell surface layer.

  The novel anchor protein and surface layer display technology according to the present invention can be applied to bacterial cell catalysts. In addition, it can be used in combination with existing techniques, and an increase in the amount of protein presented on the surface layer can be expected.

It is the electrophoresis photograph of the western blot which investigates the expression of EGFP-CBD or EGFP of an EGFP-CBD fusion expression strain and an EGFP secretion expression strain. It is a fluorescence-microscope photograph of each of proteinase K treatment and an untreated EGFP-CBD fusion expression strain and an EGFP secretion expression strain.

Claims (1)

A method of presenting a protein on the cell surface of a filamentous fungus ,
Introducing into the filamentous fungus an expression unit DNA comprising a secretion signal, a chitin binding domain, and a DNA encoding the protein to be presented; and culturing the filamentous fungus into which the expression unit DNA has been introduced, only contains the step of expressing the cell surface of said filamentous fungi,
The chitin binding domain is a peptide region comprising the amino acid sequence set forth in SEQ ID NO: 1;
The protein is fused to at least one terminal selected from the group consisting of the N-terminal side and the C-terminal side of the chitin-binding domain ;
Method.

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