JP2007189909A - New surface layer-profiling anchor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop a new surface layer-profiling anchor since the surface layer-profiling anchor is important in the surface layer-profiling engineering, and now although the anchors such as an α-agglutinin, a Flo1, etc., are developed, further excellent anchors are required. <P>SOLUTION: By paying attention to a YIL169C gene of yeast, and cloning the gene, it is found out that its sequence is different from that of published one, and a protein encoded from the newly found out sequence has a function as the surface layer-profiling anchor. Further, by deleting the N-terminal of the sequence, a more compact surface layer-profiling anchor can be prepared. The surface layer-profiling anchor is excellent in a point that it can profile the surface layer of a larger objective protein, as compared with the conventional ones. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a surface layer display anchor mainly for immobilizing a target protein on a cell surface layer among engineering technologies for displaying a target protein such as an enzyme on the surface layer of a cell.

Cell walls and membranes of cells not only serve as partition walls that maintain the structure and morphology of cells and isolate them from the outside world and other cells, but also in places such as substance recognition and permeation, information conversion and transmission, and enzyme reactions. As important. However, regarding the field called “Cell Surface Engineering”, where the surface layer of such cells is regarded as an enzyme reaction field and modified, there are several reports on its principle, There are few successful cases. Cells that have new functions by using genetic engineering techniques to present proteins localized on the cell wall or cell membrane, or a portion of them as anchors and various proteins and peptides fused to them, on the extracellular layer Can produce.
Such yeast cells are called arming yeasts, and can utilize polysaccharides and oils and fats and emit fluorescence by using cell surface engineering techniques. In addition, these surface display traits are inherited by the daughter cells of each cell, and once the surface display yeast is created, the same surface display properties are exhibited as the yeast grows. It is a technique.
At present, there are three main types of anchors that are known for the surface layer presentation.
The first is called the α-agglutinin method (GPI method). By expressing the gene encoding α-agglutinin localized on the surface of the yeast cell and the gene encoding the target protein, This technology presents the target protein at high density on the surface. The C-terminal region of α-agglutinin contains GPI (Glycosyl Phosphatidyl Inositol
) Contains anchor attachment signal. This is characterized in that it has a strong immobilization force because it is located on the C-terminal side of the target protein and is covalently bound to the cell wall β-1,6-glucan.
The second is called the Flo1 method. Flo1, a yeast aggregating protein, causes cell-to-cell adhesion and forms aggregates. This region involved in cell adhesion is used as a novel cell surface display anchor to display functional proteins on the surface of yeast cells. By using Flo1, regardless of whether it is placed at the N-terminus or C-terminus of the target protein, it exhibits immobilization ability and has a characteristic that does not depend on the domain structure of the target protein. Therefore, it was possible to display a highly active surface layer of an enzyme having an active site on the C-terminal side, which was difficult with the existing GPI anchor method. However, this is considered to act as an anchor by adsorbing to the cell wall due to a hydrophobic interaction, and has a drawback that the binding force with the cell wall is weak.
As a third group, there is a so-called PIR method. It uses yeast PIR protein, which can be covalently bound to cell wall β-1,4-glucan and can be immobilized regardless of whether it is placed at the C-terminus or N-terminus of the target protein. However, since four PIR isomers exist in yeast, it is necessary to use a yeast in which all of these isomers have been destroyed for efficient surface display. Therefore, it is difficult to put it into practical use in sake yeast, which is a diploid practical yeast.

Applied Microbiology and Biotechnology 2002 58: 291-296. (A. Kondo et al. High-level ethanol production form starch by a flocculent Saccharomyces cerevisiae strain displaying cell-surface glucoamylase.) Appl Environ Microbiol. 2002 Sep; 68 (9): 4517-22. (Matsumoto T, Fukuda H, Ueda M, Tanaka A, Kondo A. Construction of yeast strains with high cell surface lipase activity by usingnovel display systems based on the Flo1p flocculation functional domain.) Yeast. 1997 Sep 30; 13 (12): 1145-54. (Mrsa V, Seidl T, Gentzsch M, Tanner W. Specific labeling of cell wall proteins by biotinylation. Identification of four covalently linked O-mannosylated proteins of Saccharomyces cerevisiae. )

For this reason, a new surface presentation anchor for surface presentation engineering superior to the conventional ones is desired. An object of the present invention is to provide a new and more useful surface-presenting anchor.

The present inventors paid no attention to this gene because there is no detailed report on the YIL169C gene of yeast and it is registered in the database as a gene of unknown function (Saccharomyces Genome Database). In order to analyze the function of the protein encoded by this gene, cloning and determination of the nucleotide sequence were performed. As a result, it was found that, unlike the publicly available base sequence, this gene has several base substitutions, insertions and deletions near the 3 ′ end. Therefore, the estimated C-terminal amino acid sequence was different from that published on the database, and was longer at the C-terminus. As a result of investigating various properties of the newly estimated protein, it was revealed that the protein was immobilized on the cell wall and had a function as a surface layer display anchor.
Furthermore, by deleting a part of this gene, we succeeded in creating a more compact surface display anchor that is shorter than the conventional anchor.

  According to the present invention, an unprecedented surface-presenting anchor was found, and a part of the anchor was further reduced to a more compact surface-presenting anchor, and even a larger target protein could be surface-presented.

  The present invention is described in detail below.

  The present inventors focused on the yeast YIL169C gene and analyzed it in order to create a novel surface layer-presenting anchor. The entire sequence of YIL169C published by Saccharomyces Genome Database is 2988 bp and consists of 995 amino acids. At this time, when used as a surface display anchor, the region encoded by YIL169C itself is not removed except for the region necessary for surface display so that the properties of the target surface display protein are not affected, and the N-terminal side of YIL169C is 621 bp Delhi. I decided to use what I did. The base sequence is shown in SEQ ID NO: 1.

  In conducting this experiment, the above-described deletion YIL169C was actually sequenced. As a result, a part of the bases were substituted, and since there was a deletion of 3 bases and an insertion of 1 base near the 3 ′ end, the deduced amino acid sequence was 171 bp longer on the C-terminal side. In addition, the lengthened part was also missing one base compared to the publicly available chromosomal base sequence. This base sequence is shown in SEQ ID NO: 2.

  That is, the new sequence found by the present inventors was found to be a new sequence different from the published YIL169C, and this was named ANC1. As a result of investigating various properties of the protein newly deduced from the base sequence of ANC1, it was revealed that it was immobilized on the cell wall and functioned as a surface-presenting anchor. Moreover, when the amino acid sequences translated by YIL169C and ANC1 were compared, the homology rate was 90%.

Furthermore, the functional region was narrowed down to 606 bp (including the stop codon) by deleting the 5 ′ side of ANC1, which had a total length of 2535 bp (including the stop codon). This is designated as ANC3 and is shown in SEQ ID NO: 3. When the amino acid sequences translated by YIL169C and ANC3 were compared, the homology rate was 52%.
Usually, when a gene is introduced into a cell by a plasmid vector, the size of the vector that can be used is limited. Considering the units necessary for plasmid construction such as autonomous replication sequence, replication start sequence, marker gene, etc., the length of gene that can be inserted is limited, and the shorter the surface display anchor, the longer the target protein gene can be inserted. is there.
ANC3 (full length 606 bp: including a stop codon) prepared according to the present invention is shorter than generally used anchors for surface display such as α-agglutinin and Flo1, and a larger gene can be inserted.

Hereinafter, the present invention will be described in more detail.
(I) Polynucleotide The polynucleotide of the present invention comprises a highly active promoter selected from the group consisting of yeast SED1, GAPDH, PGK1, and ADH1 promoters from the 5 ′ end side, a secretion signal that functions in yeast cells. A polynucleotide (ie, an expression unit) comprising a sequence, a sequence encoding a protein of interest, and ANC1.

  A secretory signal sequence, a sequence encoding a target protein, and a fragment comprising ANC1 are linked to positions that can be expressed by a highly active promoter.

The polynucleotide may be inserted into a chromosome integration type expression vector. The structure of this expression unit inserted into a chromosomally integrated expression vector is shown in FIG.
<Highly active promoter>
In the present invention, a specific highly active promoter is used. Specific high activity promoters include yeast SED1 promoter (Japanese Patent Laid-Open No. 2003-265177), GAPDH promoter (Japanese Patent Publication No. 07-24594), PGK1 promoter (EMBO J. (1982), 1,603- Tuite, MF et al), and ADH1 It is a promoter selected from the group consisting of promoters (Nature (1981), 293, 717-Hitzeman, RA et al). These promoters exhibit high promoter activity in yeast cells.

  Among others, the present inventors have proved that the SED1 promoter, which is a promoter of the SED1 gene encoding a yeast cell wall protein, exhibits a very strong promoter activity (Japanese Patent Laid-Open No. 2003-265177).

  The yeast SED1 promoter not only exhibits high transcriptional activity under normal culture conditions, but also exhibits stable high promoter activity even under various stress environments such as high temperature, high osmotic pressure or low osmotic pressure, oligotrophic conditions, and the presence of alcohol. Show. In addition, it is not easily affected by changes in pH.

  When culturing on an industrial scale, a reaction system in which various components are dissolved, that is, a high osmotic pressure environment is often obtained. In addition, it is difficult to strictly control the temperature and pH compared to the case of culturing in a laboratory, and it is difficult to use a nutrient rich culture medium in terms of cost and the like. Thus, since the culture environment on an industrial scale is a stress environment for yeast, the polynucleotide or expression vector containing the specific highly active promoter can be suitably used for industrial substance production using recombinant yeast. It becomes.

The specific high activity promoter may be the entire region or a partial region as long as it exhibits high promoter activity.
<Secret signal sequence>
The secretory signal sequence contained in the polynucleotide of the present invention is a polynucleotide sequence encoding a secretory signal peptide.

  A secretory signal peptide is a peptide that is normally bound to the N-terminus of a secreted protein that is secreted extracellularly, including the periplasm. This peptide has a similar structure between organisms, consists of about 20 amino acids, has a basic amino acid sequence in the vicinity of the N-terminus, and then contains many hydrophobic amino acids. The secretory signal is usually removed by degradation by a signal peptidase when the secreted protein is secreted from inside the cell through the cell membrane to the outside of the cell.

In the present invention, any polynucleotide sequence encoding a secretory signal peptide capable of secreting the target protein outside the yeast cell can be used, and the origin thereof is not limited. For example, the secretion signal peptide sequence of glucoamylase such as Rhizopus oryzae, the secretion signal peptide sequence of arabinofuranosidase of Aspergillus oryzae, the α- or a-agglutinin of yeast (Saccharomyces cerevisiae) A secretory signal peptide sequence, a secretory signal peptide sequence of α-factor derived from yeast (Saccharomyces cerevisiae), and the like can be suitably used. In particular, the secretion signal peptide sequence of Rhizopus oryzae-derived glucoamylase is preferable from the viewpoint of secretion efficiency. In addition, a secretory signal peptide sequence of Aspergillus oryzae-derived glucoamylase is also preferred.
<Sequence encoding target protein>
The type of the target protein or its origin is not particularly limited. Any cDNA sequence except introns may be used. It is even better if the protein has already been expressed in yeast.
The polynucleotide sequence encoding the target protein may be a sequence encoding the entire length thereof, or may be a sequence encoding a partial region of the target protein as long as it exhibits the activity of the target protein. Moreover, as long as the activity of the target protein is exhibited, the sequence encoding the natural protein may be a sequence in which one or more nucleotides are deleted, added or substituted.
<Cell surface localized protein ANC1>
In the present invention, yeast ANC1 or a part thereof can be preferably used. ANC1 region consisting of 201 or 844 amino acids from the C-terminal side (i.e., a region consisting of 201 amino acids from the C-terminus, a region consisting of 202 amino acids from the C-terminus, ... from 843 amino acids from the C-terminus) Or a region consisting of 844 amino acids from the C-terminal), more preferably a polynucleotide sequence (ANC3) encoding a region consisting of 201 amino acids from the C-terminal side.
<Expression vector>
As described above, the polynucleotide of the present invention may be incorporated into a chromosomally integrated expression vector. An expression vector is an expression vector that is incorporated into its chromosomal DNA when introduced into yeast. As long as the expression vector does not contain an autonomously replicating sequence of yeast, it is usually a yeast chromosomal DNA integration type.

  This expression vector includes a highly active promoter and a polynucleotide fragment operably linked thereto, and this polynucleotide fragment comprises the secretory signal sequence, the target protein coding sequence, ANC1 or a part thereof.

  Any oligonucleotide sequence may be inserted between the secretory signal sequence and the target protein coding sequence as long as the secretion of the fusion protein by the secretory signal peptide is not inhibited. In general, insertion sequences up to about 30b are allowed. In addition, an arbitrary oligonucleotide sequence may be inserted between the target protein coding sequence and ANC1 as long as it is generally up to about 30b. Furthermore, an arbitrary nucleotide sequence may be inserted between the highly active promoter and the polynucleotide fragment comprising the secretory signal sequence, the target protein coding sequence, and ANC1 as long as the expression of this unit by the promoter is not inhibited. .

  The expression vector may be a plasmid vector or an artificial chromosome. A plasmid vector is preferable in that the preparation of the vector is easy and the transformation of yeast cells is easy.

  The expression vector preferably has an E. coli replication origin (ColE1) so that the expression vector can be easily constructed in E. coli. Moreover, it is usually sufficient to have a yeast selection marker (for example, drug resistance gene, URA3, TRP1, LEU2, etc.) and an E. coli selection marker (drug resistance gene, etc.). In addition to the SED1 promoter, an operator, an enhancer, a terminator, etc. may be included in order to regulate the expression of the target gene. Examples of the terminator include ADH1 (aldehyde dehydrogenase) terminator, GAPDH (glyceraldehyde 3′-phosphate dehydrogenase) terminator, and the like.

An expression vector having a SED1 promoter, which is incorporated into yeast chromosomal DNA, encodes a region consisting of 201 amino acids from the C-terminal of ANC1 to pRS406, the SED1 promoter and secretion signal sequence, the structural gene sequence of the target protein PRS406A-ANC3 constructed by inserting a polynucleotide fragment comprising the sequence (ANC3) (see Examples in the present specification) and the like.
(II) Yeast The yeast of the present invention is a yeast that holds the polynucleotide or expression vector of the present invention, and preferably this polynucleotide or expression vector is incorporated into chromosomal DNA.

  The yeast of the present invention can be obtained by introducing the polynucleotide or expression vector of the present invention into a yeast serving as a host. The introduction method is not particularly limited, and a known method can be adopted. A typical example is a method of transforming yeast using the expression vector of the present invention described above. The transformation method is not particularly limited, and known methods such as transfection methods such as calcium phosphate method, electroporation method, lipofection method, DEAE dextran method, lithium acetate method, protoplast method, and microinjection method are used without limitation. it can.

  Yeast into which the polynucleotide of the present invention has been introduced can be selected according to a conventional method using, for example, a trait by a selection marker such as URA3 or the activity of the target protein. A transformant in which a plurality of expression units derived from an expression vector are introduced can be easily obtained by selecting one having a high activity using the activity of the target protein as an index. When a drug resistance gene is used as a selection marker, a transformant having a plurality of expression vectors introduced therein can be easily selected by selecting at a drug concentration that is changed stepwise or gradually. For example, when KanMX4 is used as a selection marker, a transformant introduced with a higher copy number expression vector can be selected by using a medium having a gradient or a step in the geneticin concentration.

It can be confirmed by a conventional method that the target protein is immobilized on the cell surface layer of the obtained yeast. For example, a method in which an antibody against this protein and a fluorescently labeled secondary antibody such as FITC or an enzyme-labeled secondary antibody such as alkaline phosphatase are allowed to act on a test yeast, an antibody against this protein and a biotin-labeled secondary antibody And a method in which a fluorescent labeled streptavidin is allowed to act after the reaction.
<Host>
The yeast used as the host is not particularly limited as long as it belongs to Ascomycetou yeast. Among them, those belonging to Saccharomycetaceae are preferable, and those belonging to Saccharomyces are more preferable.

Among these, practical yeasts are preferable because they are resistant to various culture stresses and exhibit stable cell growth even in industrial production in which strict control is difficult. Practical yeasts include yeasts that are deeply involved in fermented foods, such as sake yeast, shochu yeast, wine yeast, beer yeast, and baker's yeast. Among practical yeasts, sake yeast having high fermentability and high ethanol tolerance and genetically stable is preferable.
Examples of sake yeast include “Kyokai Yeast” distributed by Japan Brewing Association and mutant strains using these as parent strains. In addition, any yeast that is used in sake brewing is useful. When an auxotrophic gene is used as a transformation marker, a strain obtained by imparting auxotrophy to these sake yeasts using a technique such as mutation may be used. Such auxotrophy is uracil auxotrophy, tryptophan. Requirement, leucine requirement, histidine requirement, adenine requirement and the like. Saccharomyces cerevisiae GRI-117-U obtained by a mutation method using “Kyokai No. 9 yeast” as a host is given as an example of imparting uracil requirement to sake yeast.
(Example)

EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated in detail, this invention is not limited to these Examples.
In the present invention, the enzyme activity was detected and the enzyme activity was measured by the following method.
<Detection of β-glucosidase activity>
The presence or absence of β-glucosidase activity was determined by adding the cells or culture supernatant to 50 mM acetate buffer (pH 5.0) containing 1 mM 4-Nitrophenyl-β-D-glucopyranoside and allowing to stand at 37 ° C. for 10 minutes. The reaction was stopped by adding an equal amount of Na ₂ CO ₃ , and the released 4-Nitrophenol was visually confirmed. Since free 4-Nitrophenol exhibits a yellow color, this coloration is confirmed.
<Detection of arabinofuranosidase activity>
The presence or absence of arabinofuranosidase activity was determined by adding the cells or culture supernatant to 50 mM acetate buffer (pH 5.0) containing 1 mM 4-Nitrophenyl α-L-arabinofuranoside and allowing to stand at 50 ° C. for 30 minutes. The released 4-Nitrophenol was visually confirmed. Since free 4-Nitrophenol exhibits a yellow color, this color is confirmed.
<Detection of glucoamylase activity>
The presence or absence of glucoamylase activity was determined using Kikkoman's “Saccharification power measurement kit”. Briefly, after adding an appropriate amount of bacterial cells or culture supernatant to an acetate buffer containing 4-Nitrophenyl β-L-maltoside, leave it at 37 ° C for 10 minutes and visually observe the released 4-Nitrophenol. Confirmed. Since free 4-Nitrophenol exhibits a yellow color, this color is confirmed.
<Detection of lipase activity>
For measurement of lipase activity, “NEFA C Test Wako” manufactured by Wako Pure Chemical Industries, Ltd. was used. Briefly, an appropriate amount of cells or culture supernatant was added to a Tris / HCl buffer solution in which tributyrin (Nacalai Tesque) was uniformly dispersed by ultrasonication, and then shaken at 37 ° C. for 16 hours. The supernatant obtained by centrifuging the reaction solution was subjected to “NEFA C Test Wako”, and the released 3-methyl-N-ethyl-N- (β-hydroxyethyl) -aniline (MEHA) was visually confirmed. Since free MEHA is bluish purple, this coloration is confirmed.

Cloning of YIL169C and determination of base sequence In accordance with a conventional method, a region containing 621 bp deletion on the 5 ′ side of YIL169C and a region containing about 200 bp on the 3 ′ side downstream was cloned. Briefly, 5'-ctcgagAGTCAATCTTCTTCCTCAGCTTCTGA-3 '(SEQ ID NO: 6) and 5'-ctcgagTGTACACATCAACCTACTCTCCCCTTCA-3' (SEQ ID NO: 7) were synthesized and used as primers for yeast Saccharomyces cerevisiae X21801A (our company's own strain) PCR was performed using the chromosomal DNA as a template. As PCR conditions, a cycle of 94 ° C./1 min-52 ° C./1 min-72 ° C./3 min was repeated 30 times. Here, restriction enzyme sites XhoI were added to the ends of both primers in order to make the ligation with other gene fragments more efficient. These additional sequences are shown in lower case in the primer sequences.
The obtained PCR product was purified by a spin column (Qiagen, PCR Purification Kit), digested with restriction enzyme XhoI, and introduced into the XhoI site of pRS406 (sold by STRATAGENE) to obtain pRS406_YIL169C. The inserted pRS406_YIL169C insert was sequenced according to a conventional method.
As a result, it was found that there were 2 base substitutions, 3 base deletions and 1 base insertion near the C-terminal. For this reason, the 3 ′ side was 171 bp longer. In addition, the lengthened portion was also missing one base compared to the published base sequence. Comparisons with published sequences are shown in FIGS.
This revealed that the C-terminus of YIL169C has a completely different structure from the published amino acid sequence. We named this new base sequence ANC1, and its base sequence is shown in SEQ ID NO: 2. The amino acid sequence translated by ANC1 is shown in SEQ ID NO: 4.

Confirmation of effect of ANC1 as surface display anchor <Preparation of DNA for displaying fluorescent protein on cell surface>
The procedure for preparing the title DNA will be described with reference to FIG.
(A) A highly active promoter (SED1 promoter: Japanese Patent Application Laid-Open No. 2003-265177) was obtained according to a conventional method. Briefly, chromosomal DNA of yeast Saccharomyces cerevisiae X21801A was used as a template using two primers 5′-cccgggGAAAAACGACAACATTCCAC-3 ′ (SEQ ID NO: 8) and 5′-gaattcCTTAATAGAGCGAACGTATT-3 ′ (SEQ ID NO: 9). PCR was performed. As PCR conditions, a cycle of 94 ° C./1 min-52 ° C./1 min-72 ° C./2 min was repeated 30 times. Here, restriction enzyme sites SmaI and EcoRI were added to the ends of both primers, respectively, in order to improve the efficiency of ligation with other gene fragments. These additional sequences are shown in lower case in the primer sequences.

The obtained PCR product was purified with a spin column (Qiagen, PCR Purification Kit) and then digested with restriction enzymes SmaI and EcoRI to obtain a SmaI-EcoRI fragment. This SmaI-EcoRI fragment contains the promoter region of the SED1 gene. The sequence is shown in SEQ ID NO: 10 of the sequence listing.
(B) A secretory signal sequence of Aspergillus oryzae glucoamylase was obtained according to a conventional method. Briefly, 5′-gaattcATGCGGAACAACCTTCTTTTT-3 ′ (SEQ ID NO: 11) and 5′-ctcgagGTTGAGATCCGACTGCCTCTT-3 ′ (SEQ ID NO: 12) were synthesized, and using these as primers, plasmids into which the glucoamylase gene was inserted PCR was performed using pNGB1 (Gene 207 127-134 (1998)) as a template. As PCR conditions, a cycle of 94 ° C./1 minute-52 ° C./1 minute-72 ° C./30 seconds was repeated 30 times. Here, restriction sites EcoRI and XhoI recognition sites were added to the design of both primers in order to increase the efficiency of ligation with other gene fragments. These additional sequences are shown in lower case in the primer sequences.

The obtained PCR product was purified with a spin column (Qiagen, PCR Purification Kit), and then cleaved with restriction enzymes EcoRI and XhoI to obtain a glucoamylase secretion signal sequence of about 100 bp. The sequence is shown in SEQ ID NO: 13 in the sequence listing.
(C) In order to obtain a sequence encoding ANC1 and its downstream region, chromosomal DNA was isolated from yeast Saccharomyces cerevisiae X21801A by a conventional method, and 5′-ctcgagAGTCAATCTTCTTCCTCAGCTTCTGA-3 ′ (SEQ ID NO: 14) and 5 PCR was performed using two primers of '-TGTACACATCAACCTACTCTCCCCTTCA-3' (SEQ ID NO: 15). As PCR conditions, a cycle of 94 ° C./1 min-52 ° C./1 min-72 ° C./3 min was repeated 30 times. Here, a restriction enzyme XhoI recognition site was added to the primer of SEQ ID NO: 14 in order to improve the efficiency of ligation with other gene fragments. These additional sequences are shown in lower case in the primer sequences.

The obtained PCR product was purified by a spin column (Qiagen, PCR Purification Kit) and digested with restriction enzymes XhoI and BsrGI to obtain an XhoI-BsrGI fragment. This XhoI-BsrGI fragment contains a sequence encoding ANC1 (844 amino acids) and 195 bp of the 3 ′ non-coding region. The sequence is shown in SEQ ID NO: 16 of the sequence listing.
(D) A fluorescent protein gene (EGFP) was obtained according to a conventional method. Briefly, 5′-ctcgagATGGTGAGCAAGGGCGAGG-3 ′ (SEQ ID NO: 17) 5′-ctcgagCTTGTACAGCTCGTCCATG-3 ′ (SEQ ID NO: 18) was synthesized, and using this as a primer, plasmid pEGFP (sold from CLONTECH) as a template PCR was performed. As PCR conditions, a cycle of 94 ° C./1 min-52 ° C./1 min-72 ° C./1 min was repeated 30 times. Here, in the design of both primers for subsequent ligation with pRS406A-ANC1, the restriction enzyme XhoI recognition site was added after removing the translation stop codon.

  The obtained PCR product was purified by a spin column (Qiagen, PCR Purification Kit) and then cleaved with a restriction enzyme XhoI to obtain a fluorescent protein sequence of about 0.7 Kbp. The sequence is shown in SEQ ID NO: 19 of the sequence listing.

The target DNA consists of the highly active promoter obtained in (A), the secretory signal sequence obtained in (B), ANC1 obtained in (C) and the fluorescent protein obtained in (D) by the following method. It is obtained by connecting with.
The SED1 promoter obtained in (A) and the secretory signal sequence obtained in (B) were ligated to the SmaI-XhoI cleavage site of pRS406 (sold by STRATAGENE) using a DNA ligase (T4 Ligase) or the like according to a conventional method. The inserted fragment was inserted. This plasmid was designated as pRS406A.
Subsequently, the XhoI-Acc65I cleavage site of pRS406A was inserted with the XhoI-BsrGI fragment containing ANC1 obtained in (C) and its downstream region to obtain plasmid pRS406A-ANC1.
Finally, the XhoI fragment of the fluorescent protein EGFP obtained in (D) was inserted into the XhoI cleavage site of pRS406A-ANC1 to obtain the desired plasmid pRS406A-EGFP-ANC1. It was confirmed by PCR that EGFP inserted into the plasmid was in the transcription direction.
<Production of yeast displaying fluorescent protein on cell surface>
Plasmid pRS406A-EGFP-ANC1 was isolated and linearized by cutting one site with the restriction enzyme StuI, and the yeast Saccharomyces cerevisiae GRI-117-U (practical sake yeast, our company's own stock, hereinafter simply GRI-117-U) and Saccharomyces cerevisiae MT8-1 (laboratory yeast, our own strain, hereinafter simply referred to as MT8-1). Culturing was carried out on an SD agar medium containing 0.67% Yeast nitrogen base w / o amino acids (Difco), 0.077% CSM-URA (BIO101) and 2% glucose, and the grown yeast was selected. In this medium, plasmid pRS406A-EGFP-ANC1 is introduced into the chromosome, and only strains with complemented uracil requirement can grow. The strains thus obtained were named GRI-117-U (EGFP-ANC1) and MT8-1 (EGFP-ANC1).

Similarly, pRS406A-EGFP was also introduced into yeast, and the resulting transformants were named GRI-117-U (EGFP) and MT8-1 (EGFP).
<Evaluation of cell surface presentation ability by fluorescence microscope observation>
GRI-117-U (EGFP-ANC1), MT8-1 (EGFP-ANC1), GRI-117-U, MT8-1 and GRI-117-U (EGFP), MT8-1 (EGFP) as comparative controls Were cultured in a liquid medium containing 0.675% Yeast nitrogen base with amino acids (SIGMA) and 2% glucose, respectively, and separated into a medium and cells by centrifugation.

  Each bacterial cell was suspended in an appropriate amount of water and then observed with a fluorescence microscope (Nicon). A part of the results is shown in FIG. The parent strains GRI-117-U and MT8-1 failed to detect a fluorescent signal. GRI-117-U (EGFP) and MT8-1 (EGFP) showed weak fluorescence signals in the cells. This was thought to be EGFP being secreted. On the other hand, GRI-117-U (EGFP-ANC1) and MT8-1 (EGFP-ANC1) showed strong fluorescence signals on the cell wall.

  From this, it became clear that it has a good cell surface presentation ability regardless of the yeast used by ANC1.

Narrowing down the region that functions as an anchor for ANC1 <Production of modified ANC1 with the N-terminal of ANC1 cut and introduction into yeast>
In order to narrow down the region that functions as an anchor for ANC1, modified ANC1 was produced by cutting out the N-terminal side of ANC1. Specifically, two primers 5′-ctcgagACTCACTGTGACGATAATGG-3 ′ (SEQ ID NO: 20) and 5′-ctcgagGTCACTTCTGAATGTCCTGA-3 ′ (SEQ ID NO: 21) were synthesized based on the internal sequence of ANC1, respectively. PCR was performed using TGTACACATCAACCTACTCTCCCCTTCA-3 ′ (SEQ ID NO: 15). As PCR conditions, a cycle of 94 ° C./1 min-52 ° C./1 min-72 ° C./1 min was repeated 30 times. Here, in the same manner as in Example 2, a restriction enzyme XhoI recognition site was added to the primers of SEQ ID NO: 20 and SEQ ID NO: 21 in order to improve the efficiency of ligation with other gene fragments. These additional sequences are shown in lower case in the primer sequences.

  The obtained PCR product was purified by a spin column (Qiagen, PCR Purification Kit) and digested with restriction enzymes XhoI and BsrGI to obtain an XhoI-BsrGI fragment. This XhoI-BsrGI fragment contains a sequence encoding 320 amino acids and 201 amino acids of a part of ANC1 and 195 bp of the 3 ′ non-coding region. The respective sequences encoding a part of ANC1 are named ANC2 and ANC3, and the respective base sequences are shown in SEQ ID NO: 22 and SEQ ID NO: 3. The amino acid sequence translated by ANC3 is shown in SEQ ID NO: 5.

  Each fragment was inserted into the XhoI-Acc65I cleavage site of pRS406A in the same manner as in Example 2 to obtain plasmids pRS406A-ANC2 and pRS406A-ANC3. Further, the fluorescent protein XhoI fragment of Example 2 was inserted into the XhoI cleavage sites of plasmids pRS406A-ANC2 and pRS406A-ANC3 to obtain the desired plasmids pRS406A-EGFP-ANC2 and pRS406A-EGFP-ANC3. It was confirmed by PCR that EGFP inserted into these plasmids was in the transcription direction.

Plasmids pRS406A-EGFP-ANC2 and pRS406A-EGFP-ANC3 were isolated, and DNA linearized by cutting one site with restriction enzyme StuI was introduced into GRI-117-U by the lithium acetate method. Culturing was carried out on an SD agar medium containing 0.67% Yeast nitrogen base w / o amino acids (Difco), 0.077% CSM-URA (BIO101) and 2% glucose, and the grown yeast was selected. In this medium, plasmids pRS406A-EGFP-ANC2 and pRS406A-EGFP-ANC3 are introduced into the chromosome, and only strains having complemented uracil requirements can grow. The strains thus obtained were named GRI-117-U (EGFP-ANC2) and GRI-117-U (EGFP-ANC3).
<Preparation of modified ANC1 with the C-terminal of ANC1 cut and introduction into yeast>
(A) In order to narrow down the region that functions as an anchor of ANC1, modified ANC1 was produced by cutting out the C-terminal side of ANC1. Specifically, primer 5′-ggtaccTCATGATTTTGGAGAAGTAGT-3 ′ (SEQ ID NO: 23) was synthesized based on the internal sequence of ANC1, and PCR was performed using 5′-ctcgagAGTCAATCTTCTTCCTCAGCTTCTGA-3 ′ (SEQ ID NO: 6). . As PCR conditions, a cycle of 94 ° C./1 min-52 ° C./1 min-72 ° C./2 min was repeated 30 times. Here, recognition sites for restriction enzymes Acc65I and XhoI were added to the two primers for efficient ligation with other gene fragments. These additional sequences are shown in lower case in the primer sequences. Further, a stop codon was added to SEQ ID NO: 23 to stop the transcription of the modified ANC1.

The obtained PCR product was purified by a spin column (Qiagen, PCR Purification Kit) and then digested with restriction enzymes XhoI and Acc65I to obtain an XhoI-Acc65I fragment. This XhoI-Acc65I fragment contains 661 amino acids excluding a part of the C-terminus of ANC1 and an artificially added stop codon (TGA). This fragment is named ANC4, and its sequence is shown in SEQ ID NO: 24.

(B) A yeast ADH1 terminator was obtained according to a conventional method. Briefly, chromosomal DNA of yeast Saccharomyces cerevisiae X21801A was used as a template using two primers, 5′-ggtaccGCGAATTTCTTATGATTTATG-3 ′ (SEQ ID NO: 25) and 5′-tgtacaGTGTGGAAGAACGATTACAAC-3 ′ (SEQ ID NO: 26). PCR was performed. As PCR conditions, a cycle of 94 ° C./1 min-52 ° C./1 min-72 ° C./1 min was repeated 30 times. Here, restriction enzyme sites Acc65I and BsrGI were added to the ends of both primers in order to make the ligation with other gene fragments more efficient. These additional sequences are shown in lower case in the primer sequences.

The obtained PCR product was purified by a spin column (Qiagen, PCR Purification Kit) and then digested with restriction enzymes Acc65I and BsrGI to obtain an Acc65I-BsrGI fragment. This Acc65I-BsrGI fragment contains the terminator region (326 bp) of the ADH1 gene. The sequence (338 bp) including the restriction enzyme site is shown in SEQ ID NO: 27.
The target DNA is obtained by connecting ANC4 obtained in (A) and the ADH1 terminator obtained in (B) and the fluorescent protein XhoI fragment produced in Example 2 by the following method.
ANC4 obtained in (A) and ADH1 terminator obtained in (B) were ligated using a DNA ligase (T4 Ligase) or the like according to a conventional method. This fragment was inserted into the XRS-Acc65I cleavage site of pRS406A to obtain plasmid pRS406A-ANC4. Further, the fluorescent protein sequence was inserted into the XhoI cleavage site of plasmid pRS406A-ANC4 to obtain the desired plasmid pRS406A-EGFP-ANC4. It was confirmed by PCR that the ADH1 terminator and EGFP inserted in the plasmid were in the transcription direction.
Plasmid pRS406A-EGFP-ANC4 was isolated, and DNA linearized by cutting one site with restriction enzyme StuI was introduced into GRI-117-U by the lithium acetate method. Culturing was carried out on an SD agar medium containing 0.67% Yeast nitrogen base w / o amino acids (Difco), 0.077% CSM-URA (BIO101) and 2% glucose, and the grown yeast was selected. In this medium, the plasmid pRS406A-EGFP-ANC4 is introduced into the chromosome, and only strains with complemented uracil requirement can grow. The strain thus obtained was named GRI-117-U (EGFP-ANC4).
<Evaluation of cell surface presentation ability by fluorescence microscope observation>
GRI-117-U (EGFP-ANC2), GRI-117-U (EGFP-ANC3) and GRI-117-U (EGFP-ANC4) are 0.675% Yeast nitrogen base with amino acids (SIGMA) and 2% glucose, respectively. Was cultured in a liquid medium containing, and centrifuged to separate the medium and cells.

  Each bacterial cell was suspended in an appropriate amount of water and then observed with a fluorescence microscope (Nicon). The results are shown in FIG. GRI-117-U (EGFP-ANC2) and GRI-117-U (EGFP-ANC3) showed a strong fluorescence signal on the cell wall, while GRI-117-U (EGFP-ANC4) showed a weak fluorescence signal in the cell. It was only observed.

  From this, it was clarified that the cell surface presentation ability exists at the C-terminal 183 amino acids of ANC1, and a very compact novel cell surface presentation anchor ANC3 containing this region was successfully obtained.

Cell surface display of β-glucosidase <Cloning of β-glucosidase gene>
(A) According to a conventional method, Aspergillus oryzae β-glucosidase cDNA was obtained. Briefly, first, total RNA was extracted from Aspergillus oryzae O-1013 strain (deposited as FERM P-16528), and then Poly (A) + RNA was obtained using oligo dT cellulose.

  Next, using Poly (A) + RNA as a template, reverse transcription reaction was performed using RT-PCR KIT manufactured by GIBCO BRL to obtain a cDNA mixture. That is, 5′-ctcgagATGAAGCTGTCAGCGGCACTTTCT-3 ′ (SEQ ID NO: 28) and 5′-ctcgagTAAGCTCAATGATCCGGTCAACTG-3 ′ (SEQ ID NO: 29) were synthesized, and PCR was carried out using these cDNA mixtures as templates. PCR was performed under the condition of repeating a cycle of 94 ° C / 1 minute-52 ° C / 1 minute-72 ° C / 2 minutes 30 times after 50 ° C / 30 minutes-94 ° C / 2 minutes. Here, in order to link with pRS406A-ANC3 produced in Example 3 later, in the design of both primers, a restriction enzyme XhoI recognition site was added after removing the translation stop codon. These additional sequences are shown in lower case in the primer sequences.

The obtained PCR product was purified with a spin column (Qiagen, PCR Purification Kit) and then cleaved with a restriction enzyme XhoI to obtain a cDNA fragment of β-glucosidase (BG1) (about 2.5 kbp). This base sequence is shown in SEQ ID NO: 30.
<Preparation of a vector that displays β-glucosidase on the surface of yeast cells>
(B) Aspergillus oryzae β-glucosidase cDNA obtained in (A) was inserted into the XhoI site of pRS406A-ANC3 prepared in Example 3 according to a conventional method to prepare the desired plasmid pRS406A-BG1-ANC3. . It was confirmed by PCR that BG1 inserted into the plasmid was in the transcription direction.
<Preparation of yeast displaying β-glucosidase on cell surface>
(C) Plasmid pRS406A-BG1-ANC3 obtained in (B) was isolated, and DNA linearized by cutting one site with restriction enzyme Sse8387I was introduced into yeast GRI-117-U by the lithium acetate method. . 0.67% Yeast nitrogen base w / o amino acids (Difco) 0.077% CSM-URA (BIO101) was cultured in an SD agar medium containing 2% glucose, and grown yeast was selected. In this medium, plasmid pRS406A-BG1-ANC3 is introduced into the chromosome, and only strains with complemented uracil requirement can grow. This yeast was named GRI-117-U (BG1-ANC3).
<Confirmation of β-glucosidase activity using surface display yeast>
(D) Yeast GRI-117-U (BG1-ANC3) obtained in (C) is cultured in YPD liquid medium containing 1% Yeast extract (Difco), 2% Polypepton (manufactured by Nippon Pharmaceutical), 2% glucose Then, the mixture was centrifuged to separate the medium and the cells, and the respective β-glucosidase activities were confirmed. At this time, GRI-117-U was used as a control.
As a result, in the control, β-glucosidase activity was not observed in the medium and the cells. On the other hand, yeast GRI-117-U (BG1-ANC3) showed β-glucosidase activity on unbroken cells.

Cell surface display of arabinofuranosidase <Cloning of arabinofuranosidase gene>
(A) Aspergillus oryzae arabinofuranosidase cDNA was obtained according to a conventional method. Briefly, first, total RNA was extracted from Aspergillus oryzae, and then Poly (A) + RNA was obtained using oligo dT cellulose.

  Next, using Poly (A) + RNA as a template, reverse transcription reaction was performed using RT-PCR KIT manufactured by GIBCO BRL to obtain a cDNA mixture. That is, 5′-ctcgagATGACTACCTTCACGAAAC-3 ′ (SEQ ID NO: 31) and 5′-ctcgagAGCCTTCCATCTCAGGAGA-3 ′ (SEQ ID NO: 32) were synthesized, and PCR was performed using this cDNA mixture as a template. PCR was performed under the conditions of repeating a cycle of 94 ° C / 1 minute-52 ° C / 1 minute-72 ° C / 2 minutes 30 times after 50 ° C / 30 minutes-94 ° C / 2 minutes. Here, in order to link with pRS406A-ANC3 produced in Example 3 later, in the design of both primers, a restriction enzyme XhoI recognition site was added after removing the translation stop codon. These additional sequences are shown in lower case in the primer sequences.

The obtained PCR product was purified by a spin column (Qiagen, PCR Purification Kit) and then cleaved with a restriction enzyme XhoI to obtain an about 1.5 kbp arabinofuranosidase cDNA fragment. The sequence is shown in SEQ ID NO: 33 of the sequence listing.
<Preparation of a vector that displays arabinofuranosidase on the surface of yeast cells>
(B) Aspergillus oryzae arabinofuranosidase cDNA obtained in (A) was inserted into the XhoI site of pRS406A-ANC3 prepared in Example 3 according to a conventional method, and the desired plasmid pRS406A-abfA-ANC3 was inserted. Produced. It was confirmed by PCR that abfA inserted into the plasmid was in the transcription direction.
<Preparation of yeast displaying arabinofuranosidase on cell surface>
(C) Plasmid pRS406A-abfA-ANC3 obtained in (B) was isolated, and DNA linearized by cutting one site with restriction enzyme StuI was introduced into yeast GRI-117-U by the lithium acetate method. . 0.67% Yeast nitrogen base w / o amino acids (Difco) 0.077% CSM-URA (BIO101) was cultured in an SD agar medium containing 2% glucose, and grown yeast was selected. In this medium, plasmid pRS406A-abfA-ANC3 is introduced into the chromosome, and only strains with complemented uracil requirement can grow. This yeast was named GRI-117-U (abfA-ANC3).
<Confirmation of arabinofuranosidase activity using surface display yeast>
(D) Yeast GRI-117-U (abfA-ANC3) obtained in (C) is cultured in YPD liquid medium containing 1% Yeast extract (Difco), 2% Polypepton (manufactured by Nippon Pharmaceutical), 2% glucose Then, the mixture was centrifuged to separate the medium and the cells, and the respective arabinofuranosidase activities were confirmed. At this time, GRI-117-U was used as a control.
As a result, the control showed no arabinofuranosidase activity in the culture medium and cells. On the other hand, yeast GRI-117-U (abfA-ANC3) showed arabinofuranosidase activity in unbroken cells.

Cell surface display of glucoamylase <Cloning of glucoamylase gene>
(A) Aspergillus oryzae glucoamylase cDNA was obtained according to a conventional method. Briefly, 5′-ctcgagCAGGCGACTGGTCTAGATACC-3 ′ (SEQ ID NO: 34) and 5′-ctcgagCCGCCAAACATCGCTCTGCAC-3 ′ (SEQ ID NO: 35) were synthesized and used as a primer as a plasmid into which a glucoamylase gene was inserted. PCR was performed using pYGA1 (Agric. Biol. Chem. 55 941-949 (1991)) as a template. As PCR conditions, a cycle of 94 ° C./1 min-52 ° C./1 min-72 ° C./2 min was repeated 30 times. Here, in order to link with pRS406A-ANC3 produced in Example 3 later, in the design of both primers, a restriction enzyme XhoI recognition site was added after removing the translation stop codon. These additional sequences are shown in lower case in the primer sequences.
The obtained PCR product was purified by a spin column (Qiagen, PCR Purification Kit) and then cleaved with a restriction enzyme XhoI to obtain an about 1.8 Kbp glucoamylase sequence. The sequence is shown in SEQ ID NO: 36 in the sequence listing.
<Preparation of a vector for presenting glucoamylase on the surface of yeast cells>
(B) The Aspergillus oryzae glucoamylase cDNA obtained in (A) was inserted into the XhoI site of pRS406A-ANC3 prepared in Example 3 according to a conventional method to prepare the desired plasmid pRS406A-glaA-ANC3. It was confirmed by PCR that glaA inserted into the plasmid was in the transcription direction.
<Production of yeast that presents glucoamylase on the cell surface>
(C) Plasmid pRS406A-glaA-ANC3 obtained in (B) was isolated and linearized by cutting one site with restriction enzyme Sse8387I and introduced into yeast GRI-117-U by the lithium acetate method. . 0.67% Yeast nitrogen base w / o amino acids (Difco) 0.077% CSM-URA (BIO101) was cultured in an SD agar medium containing 2% glucose, and grown yeast was selected. In this medium, plasmid pRS406A-glaA-ANC3 is introduced into the chromosome, and only strains with complemented uracil requirement can grow. This yeast was named GRI-117-U (glaA-ANC3).
<Confirmation of glucoamylase activity using surface display yeast>
(D) Yeast GRI-117-U (glaA-ANC3) obtained in (C) is cultured in YPD liquid medium containing 1% Yeast extract (Difco), 2% Polypepton (manufactured by Nippon Pharmaceutical), 2% glucose Then, the mixture was centrifuged to separate the medium and the cells, and the respective glucoamylase activities were confirmed. At this time, GRI-117-U was used as a control.
As a result, the control showed no glucoamylase activity in the culture medium and cells. On the other hand, yeast GRI-117-U (glaA-ANC3) showed glucoamylase activity in unbroken cells.

Cell surface display of lipase <Cloning of lipase gene>
(A) Aspergillus oryzae lipase cDNA was obtained according to a conventional method. Briefly, first, total RNA was extracted from Aspergillus oryzae, and then Poly (A) + RNA was obtained using oligo dT cellulose.

  Next, using Poly (A) + RNA as a template, reverse transcription reaction was performed using RT-PCR KIT manufactured by GIBCO BRL to obtain a cDNA mixture. That is, 5′-ctcgagATGCATCTTGCTATCAAGTCTC-3 ′ (SEQ ID NO: 37) and 5′-ctcgagGTTCGCAGCCGCAACAGCAAAT-3 ′ (SEQ ID NO: 38) were synthesized, and PCR was carried out using this cDNA mixture as a template. PCR was performed under the conditions of repeating a cycle of 94 ° C / 1 minute-52 ° C / 1 minute-72 ° C / 1 minute 30 times after 50 ° C / 30 minutes-94 ° C / 2 minutes. Here, in order to link with pRS406A-ANC3 produced in Example 3 later, in the design of both primers, a restriction enzyme XhoI recognition site was added after removing the translation stop codon. These additional sequences are shown in lower case in the primer sequences.

The obtained PCR product was purified with a spin column (Qiagen, PCR Purification Kit) and then cleaved with the restriction enzyme XhoI to obtain a lipase cDNA fragment of about 0.8 kbp. The sequence is shown in SEQ ID NO: 39 in the sequence listing.
<Preparation of a vector displaying lipase on the surface of yeast cells>
(B) The Aspergillus oryzae lipase cDNA obtained in (A) was inserted into the XhoI site of pRS406A-ANC3 prepared in Example 3 according to a standard method to prepare the desired plasmid pRS406A-tglA-ANC3. It was confirmed by PCR that tglA inserted into the plasmid was in the transcription direction.
<Production of yeast displaying lipase on cell surface>
(C) Plasmid pRS406A-tglA-ANC3 obtained in (B) was isolated, and DNA linearized by cutting one site with restriction enzyme Sse8387I was introduced into yeast GRI-117-U by the lithium acetate method. . 0.67% Yeast nitrogen base w / o amino acids (Difco) 0.077% CSM-URA (BIO101) was cultured in an SD agar medium containing 2% glucose, and grown yeast was selected. In this medium, plasmid pRS406A-tglA-ANC3 is introduced into the chromosome, and only strains with complemented uracil requirement can grow. This yeast was named GRI-117-U (tglA-ANC3).
<Confirmation of lipase activity using surface display yeast>
(D) Yeast GRI-117-U (tglA-ANC3) obtained in (C) is cultured in YPD liquid medium containing 1% Yeast extract (Difco), 2% Polypepton (manufactured by Nippon Pharmaceutical), 2% glucose Then, the mixture was centrifuged to separate the medium and the cells, and the respective lipase activities were confirmed. At this time, GRI-117-U was used as a control.
As a result, no lipase activity was observed in the medium and the cells of the control. On the other hand, yeast GRI-117-U (tglA-ANC3) showed lipase activity on unbroken cells.

It is a figure which shows the expression cassette structure for displaying a target protein on a cell surface layer. This is a comparison of the public sequence of YIL169C and the base sequence of ANC1. The same as above. The same as above. The same as above. The same as above. The same as above. It is the figure which showed the production method of the vector which evaluates the surface layer display anchor ability of ANC1. It is a fluorescence-microscope observation image of the result of having evaluated the surface layer presentation anchor ability of ANC1. It is the fluorescence microscope observation image of the result which narrowed down the surface layer display anchor ability of ANC1.

Claims (7)

A novel surface-presented anchor protein having the amino acid sequence shown in SEQ ID NO: 4 in the Sequence Listing. A novel surface-presenting anchor protein having the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing. The DNA encoding the protein according to claim 1, which is represented by the nucleotide sequence of SEQ ID NO: 2. The DNA encoding the protein according to claim 2, which is represented by the nucleotide sequence of SEQ ID NO: 3. A surface layer display protein in which the protein according to claim 1 or 2 is connected to the C-terminal side of the target protein. A plasmid vector wherein the DNA according to claim 3 or 4 is connected to the C-terminal side of the target protein in a form capable of being expressed as a fusion protein. A transformant of yeast Saccharomyces cerevisiae comprising introducing the plasmid vector according to claim 6.

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