JP2016158544A - Novel protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To find a novel protein with a silica formation activity and to produce silica and a protein that is immobilized to silica using the same.SOLUTION: The invention relates to a production method of a protein with silica formation activity, the protein being characterized by hydrofluorination and subsequent extraction of the structure of Hyalospongia, as well as to a protein with silica formation activity, wherein the molecular weight measured by SDS polyacrylamide gel electrophoresis is 23 kDa, the optimum pH of silica formation is 6-8, and Lys His Asp His His Asp His His His Ser His Asp Pro is produced as an amino acid sequence of a trypsin digestion fragment.SELECTED DRAWING: None

Description

  The present invention relates to a novel protein. Specifically, the present invention relates to a novel protein having silica forming activity.

  Silica is silicon dioxide or a material composed thereof. It is the most abundant substance in the crust, and some are produced by organisms such as diatoms and sponges. Silica is used in many fields. For example, silica gel obtained from silica as a raw material is used as a desiccant in pharmaceuticals, foods, and the like. Silica is also widely used as a filter aid. In addition, quartz glass, silica gel, aerogel, etc. are used in a wide range of fields such as construction, electronics, and materials as building materials, insulators, abrasives, fillers, antifoaming agents, and additives.

  Silica is collected in large quantities from soil. Used as is or further processed. The industrial process is heating silicon tetrachloride at a high temperature of 3000 ° C., neutralizing alkali silicates with sulfuric acid, heating silicate esters or hydrolysis with acids and alkalis, both of which affect the environment. Effect. Although there is a report that silica was generated by an enzymatic reaction, mass production has not been achieved.

  Silica produced by living organisms contains trace amounts of organic substances, which have been shown to be involved in silica production. Silafine, a peptide rich in lysine and serine, was found in the silica skeleton of diatoms, and it was confirmed that the peptide promotes silica formation (Non-Patent Document 1). Silafine has a complex structure in which the lysine residue is modified with polyamine and the serine residue is phosphorylated, and its production process is complicated by synthesizing the peptide backbone and then applying two types of scientific modifications. is there. A silica-forming enzyme silicatein has been found from the silica skeleton of sponges (Non-patent Document 2). Since silicatein is a protein belonging to the papain-like cysteine protease superfamily and its activity is lost by heat treatment, a higher order structure is essential. As described above, there are many problems in using silafine and silicatein and it is difficult to put it to practical use.

  Among sponge animals, six-release sponges produce silica as glass fibers, have the same structure as optical fibers, and have the property of actually transmitting light (Non-patent Document 3). Moreover, sponge glass fibers are thinner than optical fibers and are superior in strength. If silica having such unique and excellent physical properties can be produced at a low temperature by the action of a biocatalyst, a method for producing silica that is environmentally friendly and advantageous in terms of energy can be established.

Nils Kroeger et al. Science, Vol.286, pp.1129-1132 (1999) Katsuhiko Shimizu et al. Proc. Natl. Acad. Sci. USA, Vol.95, pp.6234-6238 (1998) Vikram C. Sundar et al. Nature, Vol.424, pp.899-900 (2003)

  The problem to be solved by the invention is to find a new protein having silica-forming activity, to identify the nucleotide sequence and amino acid sequence thereof, and to obtain such protein by a recombinant method, and to convert silica having excellent physical properties into energy. It is also advantageous for mass production under low temperature conditions.

  The present inventors have intensively studied to solve the above-mentioned problems, and found a novel protein that promotes silica formation at pH 6 to 8 in the silica skeleton of glass sponges Kairodoketsu and Hosugai, and completed the present invention. I came to let you. The present inventors named this protein glassine (in the present specification and drawings, the protein having silica-forming activity of the present invention may be referred to as glassine).

Accordingly, the present invention provides the following:
(1) A method for producing a protein having silica-forming activity, wherein the protein is extracted from a glass sponge skeleton.
(2) A protein having silica-forming activity obtained by the method described in (1), having a molecular weight measured by SDS polyacrylamide gel electrophoresis of 22 kDa to 24 kDa, and an optimum pH for silica formation of 6 to 8 A protein that produces a peptide consisting of Lys His Asp His His As His His Ser Ala Pro (SEQ ID NO: 1) as the amino acid sequence of a trypsin digested fragment.
(3) The following nucleotide sequence:
(A) the nucleotide sequence of positions 55 to 702 of SEQ ID NO: 10;
(B) a nucleotide sequence having 90% or more nucleotide sequence identity to the nucleotide sequence of (a),
(C) A polynucleotide comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of (a) or (b), and encoding a protein having silica-forming activity.
(4) A protein having silica-forming activity encoded by the polynucleotide according to (3).
(5) The following amino acid sequence:
(A) the amino acid sequence of positions 19 to 233 of SEQ ID NO: 11,
(B) an amino acid sequence having 90% or more amino acid sequence identity to the amino acid sequence of (a),
(C) A protein having silica-forming activity, comprising any amino acid sequence in which 1 to about 40 amino acids are substituted, deleted or added in the amino acid sequence of (a) or (b).
(6) A method for producing silica, comprising reacting the protein according to any one of (2), (4) and (5) with silicic acid.
(7) A fusion protein of the protein according to any one of (2), (4) and (5) and a target protein is prepared, and then the fusion protein is reacted with silicic acid, which is immobilized on silica. Method for producing a target protein.
(8) A vector that expresses a protein having silica-forming activity, comprising the polynucleotide according to (3).
(9) A method for producing a protein having silica-forming activity, wherein the vector according to (8) is introduced into a host to express a protein having silica-forming activity.

  By using the novel protein glassine of the present invention, silica and a protein immobilized on silica can be easily obtained. Furthermore, according to the present invention, glassine can be produced by a recombinant method, and mass production of silica becomes possible. The method for producing silica according to the present invention can be carried out at a low temperature, and is an energy-friendly and environmentally friendly method.

FIG. 1 is a photograph of an electrophoretic gel showing the extraction results of glassine from the silica skeletons of kaidouroketsu (Euplectella aspergillum, lane 1) and Euplectella sp. (Lane 2). FIG. 2 is a graph showing the results of examining the silica-forming ability (left panel) and optimum pH (right panel) of glassine. FIG. 3 is a photograph of silica accumulated at the bottom of the tube when silica is produced from silicic acid using glassine in an Eppendorf tube. FIG. 4 is a scheme of a glassine expression vector. FIG. 5 is a graph showing the silica-forming activity of glassine obtained by the recombinant method. The left bar shows natural glassine and the right bar shows recombinant glassine.

  In one aspect, the present invention provides a method for producing a protein having silica-forming activity, wherein the protein is extracted from a skeleton of a glass sponge. Glass sponges, also called hexagonal sponges, are sponges having a skeleton with bone needles made of 4 or 6 silica. Glass sponges are classified into two subclasses (both board subclass and six-star subclass) and five eyes (both board, Aulocalycodia, sixth release, Lychniscosida, and Cidouridae). The types of sponges belonging to these eyes are known. For example, the sponge of the family Hosugidae is included in both boards, and the sponge of the family Cagerosceae is included in the order of Kairoudokesu. Examples of the Kairoudaceae include species such as Kairoudoketsu, Owen Kairoudoketsu, Yamato Kairoudoketsu, Marshall Kairoudoketsu.

Organic substances adhering to the skeleton of the glass sponge are removed using an aqueous sodium hypochlorite solution, and the obtained skeleton is treated with hydrogen fluoride (for example, an HF / NH ₄ F aqueous solution) to obtain a silica solution. The silica solution is dialyzed to obtain a dialyzed solution, and if necessary, glassine can be extracted from glass sponges by concentrating the dialyzed solution by means such as ultrafiltration. It goes without saying that the extraction method of glassine is not limited to the above method.

  The protein (glassine) having silica-forming activity of the present invention has silica-forming activity. The silica forming activity is an activity for forming silica from silicic acid. The silica forming activity can be measured, for example, by adding glassine to a supersaturated silicic acid aqueous solution and quantifying the generated silica precipitate.

  Glassine derived from glass sponge skeleton has a molecular weight measured by SDS polyacrylamide gel electrophoresis of about 20 to about 26 kDa, typically about 22 kDa to 24 kDa, for example 23 kDa, and has an optimum pH for silica formation. A peptide consisting of about 5 to about 9, typically about 6 to about 8, and consisting of Lys His Asp His His His His His Ala Pro (SEQ ID NO: 1) In the amino acid sequence of No. 1, a peptide consisting of an amino acid sequence in which 1, 2, 3, 4 or 5 amino acid residues are substituted, added or deleted, or the amino acid sequence of SEQ ID No. 1 60% or more, preferably 70% or more, more preferably 80% or more, even more preferred Ku can also be defined as a protein that produces a peptide consisting of an amino acid sequence having at least 90% identity.

The invention in a further aspect provides the following nucleotide sequence:
(A) the nucleotide sequence of positions 55 to 702 of SEQ ID NO: 10;
(B) a nucleotide sequence having 90% or more nucleotide sequence identity to the nucleotide sequence of (a),
(C) a polynucleotide comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of (a) or (b), the polynucleotide encoding a protein having silica-forming activity Is.

  A typical example of a nucleotide sequence encoding glassine of the present invention is a nucleotide sequence consisting of nucleotide sequences 55 to 702 of the nucleotide sequence shown in SEQ ID NO: 10, or 55 of the nucleotide sequence shown in SEQ ID NO: 10. It is a nucleotide sequence comprising the nucleotide sequence from position 702. A protein (glassine) encoded by such a nucleotide sequence has silica-forming activity.

  The glassine of the present invention may be encoded by a mutated sequence of the above nucleotide sequence. Examples of the mutant sequence of the nucleotide sequence include those having substantial nucleotide sequence identity to the nucleotide sequence at positions 55 to 702 of the nucleotide sequence shown in SEQ ID NO: 10. As used herein, “having substantial nucleotide sequence identity” means 80% or more, preferably 90% or more, more preferably 95% when a plurality of (for example, two) nucleotide sequences are compared. More preferably, it means having a nucleotide sequence identity of 96%, 97%, 98% or 99% or more. In addition,% of identity means the value computed using well-known software (for example, FASTA, BLAST etc.) which calculates the identity between several (for example, two) nucleotide sequences by default setting. As a further example of the above-mentioned mutant nucleotide sequence, 1 to about 60 nucleotides are added to the 5 ′ end of the nucleotide sequence of positions 55 to 702 of the nucleotide sequence shown in SEQ ID NO: 10, and 1 is added to the 3 ′ end. Examples include a nucleotide sequence to which about 30 nucleotides are added, and a specific example thereof is the nucleotide sequence shown in SEQ ID NO: 10. Proteins (glassin) encoded by these mutated nucleotide sequences have silica-forming activity.

  As a further example of the mutated sequence of the above nucleotide sequence, a nucleotide sequence comprising a nucleotide sequence that hybridizes under stringent hybridization conditions to the nucleotide sequence encoding glassine described above, or consisting of such a nucleotide sequence Nucleotide sequences are mentioned. The “stringent conditions” used in the present specification are not limited to these, but include, for example, the following hybridization and washing conditions. As hybridization conditions, washing conditions are 30 to 50 ° C., 3 to 4 × SSC (150 mM sodium chloride, 15 mM sodium citrate, pH 7.2), 0.1 to 0.5% SDS for 1 to 24 hours. Can be an operation at room temperature with a solution containing 0.2 × SSC and 0.1% SDS at room temperature. However, the combinations of the above conditions are exemplary, and those skilled in the art will understand the above or other factors that determine the stringency of hybridization (for example, hybridization probe concentration, length and GC content, hybridization reaction). It is possible to achieve the same stringency as above by appropriately combining the time and the like. Proteins (glassin) encoded by these mutated nucleotide sequences have silica-forming activity.

In a further aspect, the present invention provides the following amino acid sequence:
(A) the amino acid sequence of positions 19 to 233 of SEQ ID NO: 11,
(B) an amino acid sequence having 90% or more amino acid sequence identity to the amino acid sequence of (a),
(C) The present invention relates to a protein having silica-forming activity, comprising any amino acid sequence in which 1 to about 40 amino acids are substituted, deleted or added in the amino acid sequence of (a) or (b).

  The silica-forming protein (Glasin) of the present invention typically comprises the amino acid sequence of positions 19 to 233 of SEQ ID NO: 11, or the amino acid sequence of positions 19 to 233 of SEQ ID NO: 11. It is a protein consisting of

  The glassine of the present invention may be a mutant of the above glassine. Such a mutant may contain a mutant sequence of the above amino acid sequence, or may comprise a mutant sequence of the above amino acid sequence. Examples of the mutated sequence of the amino acid sequence include those having substantial amino acid sequence identity to the amino acid sequence at positions 19 to 233 of SEQ ID NO: 11. As used herein, “having substantial amino acid sequence identity” means 80% or more, preferably 90% or more, more preferably 95% when a plurality of (for example, two) amino acid sequences are compared. More preferably, it means having 96%, 97%, 98% or 99% or more amino acid sequence identity. In addition,% of identity means the value calculated using well-known software (for example, FASTA, BLAST etc.) which calculates the identity between several (for example, two) amino acid sequences by default setting. The glassine of the present invention containing such a mutated amino acid sequence or consisting of such a mutated amino acid sequence has silica-forming activity.

  Examples of further mutated sequences of the above amino acid sequence include 1 to 40, preferably 1 to 30, more preferably 1 to 20, in the amino acid sequence of positions 19 to 233 of SEQ ID NO: 11. More preferably, one to several amino acid sequences (for example 1, 2, 3, 4, 5, 6, 7, 8, 9) are substituted, deleted or added. Is exemplified. As an example of the above mutant amino acid sequence, 1 to about 20 amino acids are added to the N-terminus of the amino acid sequence of positions 19 to 233 of the amino acid sequence shown in SEQ ID NO: 11, and 1 to about 10 are added to the C-terminus. An amino acid sequence to which an amino acid is added is mentioned, and a specific example thereof is the amino acid sequence shown in SEQ ID NO: 11. A mutant of glassine consisting of or containing such a mutated amino acid sequence also has silica-forming activity. The substitution, deletion or addition of amino acids in the amino acid sequence can be performed by methods known to those skilled in the art. These glassine variants also have silica-forming activity.

  Furthermore, as a mutant of glassine of the present invention, (a) the amino acid sequence of positions 19 to 109 of SEQ ID NO: 11, (b) 80% or more, preferably 85% or more with respect to the amino acid sequence of (a), More preferably 90% or more, still more preferably 95% or more, for example 96% or more, 97% or more, 98% or more, 99% or more of the amino acid sequence, or (c) (a) or (b) A protein comprising or comprising an amino acid sequence in which 1 to about 20, preferably 1 to several amino acids are substituted, deleted or added in the amino acid sequence, and having a silica-forming activity Is mentioned. In addition, as a mutant of glassine of the present invention, (d) an amino acid sequence at positions 132 to 227 of SEQ ID NO: 11, 80% or more, preferably 85% with respect to the amino acid sequence of (e) (d) Or more, more preferably 90% or more, still more preferably 95% or more, for example, 96% or more, 97% or more, 98% or more, 99% or more amino acid sequence, or (f) (d) or (e A protein comprising or including an amino acid sequence in which 1 to about 20, preferably 1 to several amino acids have been substituted, deleted or added, and having a silica-forming activity. It is done.

  In a further aspect, the present invention provides a method for producing silica, characterized by reacting glassine or a glassine variant with silicic acid. The method can be performed, for example, by mixing a silicic acid solution and an aqueous glassine solution or an aqueous glassine mutant solution, and incubating at an optimum pH and temperature of the enzyme to form a silica precipitate. See the examples for specific examples of reaction conditions.

  In a further aspect, the present invention provides a production of a target protein immobilized on silica, characterized in that a fusion protein of glassine or a glassine variant and a target protein is prepared, and then the fusion protein and silicic acid are reacted. Provide a method. The target protein may be any protein that is immobilized on silica as an immobilization carrier. Methods for producing fusion proteins are known to those skilled in the art. In this method of the present invention, the target protein immobilized on silica can be easily obtained simply by reacting the fusion protein with silicic acid.

  In a further aspect, the present invention provides a vector comprising a polynucleotide encoding glassine or a variant thereof, wherein the vector expresses a protein having silica-forming activity (glassin). Such a vector may be obtained by incorporating a polynucleotide encoding glassine or a variant thereof into a known expression vector. Methods for incorporating polynucleotides into expression vectors are also known to those skilled in the art.

  In a further aspect, the present invention provides a method for producing a protein having silica-forming activity, wherein the vector is introduced into a host to express a protein having silica-forming activity. As the host, known cells such as bacterial cells, animal cells, and insect cells can be used. Methods for introducing vectors into cells are also known to those skilled in the art. It is also possible to mass-produce glassine or a variant thereof using such a method.

  The present invention will be described in more detail and specifically with reference to the following examples. However, the examples do not limit the scope of the present invention.

Example 1. Isolation of glassine from coconut butter and determination of its gene sequence 1) Materials The skeleton of Euplectella aspergillum was obtained from the Philippines and used. Euplectella sp. Living organisms and skeletons of Hyalonema sieboldi were collected by beam trawling in the East China Sea off Fukue City.

2) Extraction of glassine In order to remove organic substances adhering to the skeleton of Euplectella aspergillum, they were immersed in a 0.1% aqueous sodium hypochlorite solution overnight and then washed with distilled water. 2 g of skeleton was immersed in 50 mL of a 2M HF / 8M NH ₄ F aqueous solution to dissolve silica. The solution was dialyzed against 10 volumes of water and the external solution was changed 7 times every 5 to 10 hours. Thereafter, the dialysis internal solution was recovered, and a water-soluble fraction was obtained by centrifugation at 10,000 × g for 20 minutes. Further, the water-soluble fraction was concentrated to 0.2 mL by ultrafiltration (Amicon Ultra-15 Ultracel (3,000 NMWL), Millipore). Protein was quantified using DC protein assay kit (Bio-Rad, USA) to obtain a 1 mg / mL water-soluble fraction.

3) Identification of glassine When 1 μg of protein in water-soluble fraction was developed by SDS polyacrylamide gel electrophoresis and visualized by Coomassie brilliant blue staining, a 23 kDa protein was recognized as the main protein, and almost all other proteins were observed. None (Figure 1). The 23 kDa protein is the main component of the water-soluble protein contained in the skeleton of the Kairoudoketsu, this protein is named glassin, and the water-soluble fraction is called glassine aqueous solution. Similar results were obtained in water-soluble fractions extracted from Euplectella sp. And Hosugai skeletons.

  For glassine from Euplectella aspergillum and Euplectella sp., Lys His Asp His Asp His His Asp His Ala Pro was obtained as the amino acid sequence of the trypsin digested fragment.

4) Acceleration of silica formation Glacine is dissolved in 100 mM sodium phosphate buffer (pH 4, 5, 6, 7 or 8) to give glassine solution (0.5, 1, 2.5, 5 or 12.5 μg / ul). Was prepared. 1 μL of a supersaturated silicic acid solution prepared by stirring a mixed solution of 15 μL of tetramethoxysilane and 85 μL of 1 mM hydrochloric acid for 5 minutes was added to the glassine solution, and the precipitate was collected by centrifugation after 5 minutes. As a control experiment, the same operation was carried out using only the buffer solution, but no precipitate was recovered at any pH. The precipitate was washed twice with water. The precipitate was dissolved in 0.1 M NaOH to quantify silicic acid (Iler RK (1979) The chemistry of silica: solubility, polymerization, colloid and surface properties, and biochemistry (Wiley, New York). Performed in accordance). As shown in FIG. 2, silica precipitation increased in proportion to glassine concentration at pH 6 (left panel in FIG. 2). Silica precipitation was observed at pH 6-8 (relative activity of 50% or more) and was not observed at pH 4-5 (FIG. 2, right panel). The morphology was observed with a scanning electron microscope (FIG. 3). It was found that glassine promotes silica formation at pH 6-8. The above properties were common to glassine of Euplectella aspergillum and Euplectella sp.

5) Gene isolation cDNA was synthesized by using RNA reverse transcriptase using RNA extracted from Owen Kairoud tissue as a template and oligo dT-3sites adaptor (Takara Bio) as a primer. Primer 5'-ar kay kay kay kay kay c-3 '(primer 1) (SEQ ID NO: 2) and 5'- kay kay kay kay gcn c-3 against the DNA sequence assumed from the partial amino acid sequence of glassine '(Primer 2) (SEQ ID NO: 3) was designed. The cDNA fragment RT-PCR1 was amplified by PCR using Taq polymerase using primer 1 and a partial sequence of oligo dT-3sites adaptor as primers with the cDNA synthesized as described above as a template. The amplified cDNA fragment was inserted into the pCR4 vector and cloned, and the base sequence was determined (SEQ ID NO: 4). A part of the obtained sequence was used as a primer to amplify the 5 ′ portion by the 5′-RACE method. As a result, three types of clones (SEQ ID NOs: 5, 6, 7) were obtained. 5′-RACE was performed again using a sequence common to the three types of clones (5′-tggttttctctgtgtccactac-3 ′ (SEQ ID NO: 8)) to obtain 5′-RACE2 / clone 1 (SEQ ID NO: 9). . Thus, a cDNA sequence encoding glassine was obtained. It was confirmed that this sequence was amplified by the PCR method, and the resulting clone was named RT-PCR2. The nucleotide sequence of RT-PCR2 is shown in SEQ ID NO: 10, and the amino acid sequence encoded by it is shown in SEQ ID NO: 11. The amino acid sequence of the trypsin digestion fragment already obtained is not Lys His Asp His His As His His His Asp His Ala Pro but Lys His Asp His His As His His Ala Pro (SEQ ID NO: 1) There was found.

Example 2 Production of recombinant glassine 6) Production of recombinant protein Part of RT-PCR2 (positions 55 to 702 of SEQ ID NO: 10) was inserted into pET100 to construct a glassine expression vector (FIG. 4). This vector was introduced into Escherichia coli BL2 Star (DE3) strain and cultured in a medium supplemented with 0.1 mM IPTG at 37 ° C. for 4 hours to recover the cells. The cells were lysed with Fastbreak cell lysis reagent (Promega), and recombinant glassine was purified with a His-tag protein affinity column (HisGraviTrap, GE, USA). Recombinant glassine showed silica-forming activity equivalent to natural glassine (FIG. 5).

  Artificial mutation was introduced into the DNA sequence of RT-PCR2 (the sequence of the encoded protein was not affected), and it was confirmed that the recombinant glassine produced using this mutant sequence had silica synthesis activity. (Data not shown).

  The present invention can be used in a wide range of fields such as cosmetics, pharmaceuticals, and foods.

Claims (9)

  A method for producing a protein having silica-forming activity, characterized by being extracted from a skeleton of glass sponges.   A protein having silica-forming activity obtained by the method according to claim 1, wherein the molecular weight measured by SDS polyacrylamide gel electrophoresis is 22 kDa to 24 kDa, and the optimum pH for silica formation is 6 to 8, A protein that produces a peptide consisting of Lys His Asp His His As His His Ser Ala Pro (SEQ ID NO: 1) as the amino acid sequence of a trypsin digested fragment. The following nucleotide sequence:
(A) the nucleotide sequence of positions 55 to 702 of SEQ ID NO: 10;
(B) a nucleotide sequence having 90% or more nucleotide sequence identity to the nucleotide sequence of (a),
(C) A polynucleotide comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of (a) or (b), and encoding a protein having silica-forming activity.   A protein having silica-forming activity encoded by the polynucleotide of claim 3. The following amino acid sequence:
(A) the amino acid sequence of positions 19 to 233 of SEQ ID NO: 11,
(B) an amino acid sequence having 90% or more amino acid sequence identity to the amino acid sequence of (a),
(C) A protein having silica-forming activity, comprising any amino acid sequence in which one to several amino acids are substituted, deleted or added in the amino acid sequence of (a) or (b).   A method for producing silica, comprising reacting the protein according to any one of claims 2, 4, and 5 with silicic acid. A production of a protein immobilized on silica, characterized in that a fusion protein of the protein according to any one of claims 2, 4 and 5 and a target protein is prepared and then the fusion protein and silicic acid are reacted. Method.   A vector for expressing a protein having silica-forming activity, comprising the polynucleotide according to claim 3.   A method for producing a protein having silica-forming activity, wherein the vector according to claim 8 is introduced into a host to express a protein having silica-forming activity.