JP2006197825A - Method for producing silica microparticle using eschelichia coli - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique for easily obtaining silica microparticles at low cost based on the biomineralization of Bacillariophyta(diatom). <P>SOLUTION: A method for producing the silica microparticles is provided, comprising the step of culturing, using a silisic acid-containing medium, Eschelichia coli transformed with an expression vector containing the base sequence mentioned below(Sequence No.1) as a gene encoding a peptide to be expressed and the sequence complementary thereto. Preferably, the silicic acid-containing medium to be used is prepared by adding tetramethoxysilane and dilute hydrochloric acid, or by using water with diatom alive therein. The base sequence(Sequence No.1) is ATG AGC AGC AAA AAA AGC GGC AGC TAT TCT GGC TCT AAA GGT AGT AAA CGT CGC ATT TTG TAA. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention belongs to an application field of biomineralization, and particularly relates to a novel technique for producing silica fine particles using a genetic engineering technique.

Living organisms can make various mineral structures with fine processing, and this action is called biomineralization.
For example, diatoms, which are algae that are widely distributed in large quantities in nature, have their cell surfaces covered with silica, which precipitates silica when several types of proteins (polypeptides) act in concert. I know that is to make it happen. One such polypeptide, silaffin, contains many cationic amino acids and is believed to promote the concentration of silicon anions and the precipitation of silica. For example, silafine 1A (sil1A) is a polypeptide that is highly modified by phosphorylation or the like, and is known to produce nanoscale silica fine particles (nanosilica spheres) from silicic acid under weak acidity [for example, N. Kroeger et al., J. Biol.
Chem., 276, 26066-26070 (2001): Non-patent document 1].

For silafine 1A, a chemically synthesized unmodified R5 peptide partial peptide of silafin 1A is known to produce fine silica particles within a few minutes at room temperature under neutral pH when mixed with silicic acid. [For example, N. Kroeger et al., Science, 286,
1129-1132 (1999): Non-Patent Document 2]. Furthermore, it has also been reported that silica particles can be produced without the need for post-translational modification even under weak basicity using R5 peptide [N. Kroeger et al., Scinece, 298,
584-586 (2002): Non-patent document 3].

Thus, attempts have been made to produce silica fine particles by utilizing the above phenomenon. To date, the methods used with silafine are silafine proteins purified from cultured silicon, or chemically synthesized silafine. There are only those using 1A polypeptide. Purification from diatoms has very special properties such as silafin protein bound to the silica surface, which requires a very complicated process, and culturing diatoms is often difficult. In addition, chemical synthesis is very disadvantageous in terms of cost in the case of mass production such as increasing the scale.
N. Kroeger et al., J. Biol. Chem., 276, 26066-26070 (2001) N. Kroeger et al., Science, 286, 1129-1132 (1999) N. Kroeger et al., Scinece, 298, 584-586 (2002)

  An object of the present invention is to provide a new technique based on biomineralization of diatoms and capable of obtaining silica fine particles (nanoscale silica particles) easily and at low cost.

  As a result of repeated studies, the present inventor succeeded in constructing an expression system capable of producing the R5 peptide in Escherichia coli by genetic engineering techniques, and derived the present invention by utilizing this for silica synthesis. is there.

  Thus, according to the present invention, Escherichia coli transformed with an expression vector containing the following base sequence (SEQ ID NO: 1) and a complementary sequence thereof as a gene encoding a peptide to be expressed is obtained using a silicate-containing medium. There is provided a method for producing silica fine particles, which comprises a step of culturing.

According to a preferred embodiment of the present invention, a silicic acid-containing medium prepared by adding tetramethoxysilane and dilute hydrochloric acid is used. According to another preferred embodiment of the present invention, a silicate-containing medium prepared using water in which diatoms are alive is used.
Furthermore, the present invention provides, as another aspect, E. coli transformed with an expression vector comprising the base sequence consisting of SEQ ID NO: 1 and a complementary sequence thereof.

  According to the present invention, silica fine particles can be produced by culturing Escherichia coli that can be carried out relatively easily according to a conventional method. The reaction of producing silica fine particles automatically proceeds when a raw material of silica is put in a culture solution (medium). In particular, the present invention is used for water in which diatoms live as a silica source (for example, existing It is very advantageous in terms of cost in that the water of the pond can be used.

  It is known that the partial peptide of silafine 1A called R5 peptide described above is composed of 19 amino acids of the sequence (SSKKSGSYSGSKSKRRIL: SEQ ID NO: 2) (for example, Non-Patent Document 2). The base sequence represented by SEQ ID NO: 1 contained in the expression vector used in the present invention is designed by the present inventor so that the R5 peptide can be expressed specifically in E. coli, and the R5 peptide of diatom itself. Is different from the nucleotide sequence (gene sequence).

  In order to carry out the method for producing silica fine particles of the present invention, an expression vector comprising the base sequence of SEQ ID NO: 1 and a complementary sequence thereof is constructed as a gene encoding a peptide to be expressed (R5 peptide). The transformed E. coli is cultured using a silicate-containing medium. Here, techniques such as construction of expression vectors, transformation, and culture of E. coli may be performed by appropriately employing operations and materials well known in the art as described below.

  For example, in order to construct an expression vector, a DNA fragment containing a double-stranded DNA consisting of the nucleotide sequence of SEQ ID NO: 1 and its complementary sequence is amplified with a cloning vector and then inserted into another vector to express the expression vector. And

  In order to obtain a double-stranded DNA fragment as a raw material, an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 1 and an oligonucleotide containing a sequence complementary to the nucleotide sequence of SEQ ID NO: 1 may be synthesized with a DNA synthesizer. Actually, it is not necessary to synthesize the full length of each oligonucleotide. After synthesizing a part of each oligonucleotide so that the complementary parts of the two oligonucleotides overlap, If provided, a desired double-stranded DNA can be obtained.

  In addition, the cloning vector used in the present invention includes (1) a selection marker for discriminating between a host cell (E. coli) holding the vector and a cell not holding it, and (2) restrictions for incorporating the target DNA fragment. Any one can be used without particular limitation as long as it has an enzyme site and (3) has the ability to replicate a large number of itself in a host cell (E. coli). For example, Bluescript II KS (+) [Toyobo Co., Ltd.] can be mentioned as a suitable cloning vector for use in the present invention. Since this vector has a number of restriction enzyme sites in the β-galactosidase gene, color selection (distinguishes the presence or absence of foreign DNA from the colony's white color and blue color), and selection by antibiotic resistance is also possible. Is known.

  In the present invention, various vectors including a promoter and a terminator can be used as the origin vector of the expression vector, and those including an inducible promoter are particularly preferable. From this point, a pET vector such as pET30c (Takara Bio Inc.) is particularly preferable as the origin vector of the expression vector in the present invention. This vector can mass-produce the target protein (oligopeptide) by adding an inducer IPTG (isopropyl 1-thio-β-D-galactoside). The restriction enzyme site for incorporating a foreign gene (DNA) in this vector is basically BamHI / Hind III, and the target oligopeptide is generally expressed as a histidine tag fusion, but Nde I can be used. For example, it can be expressed as a normal oligopeptide starting from the initiation codon.

  What is necessary is just to select suitably the kind of colon_bacillus | E._coli suitable for using in this invention according to the kind of vector to be used. For example, it is preferable to use DH5α [Toyobo Co., Ltd.], which has a high transformation efficiency and can perform color selection, for the preparation of the above-described vector derived from Bluescript II KS (+). Examples of suitable Escherichia coli for expressing a desired oligopeptide (R5 peptide) after being transformed with the vector derived from BL21 (DE3) pLysS [Takara Bio Inc.].

  Cultivation of E. coli for preparing a cloning vector and an expression vector may be performed according to a technique known in the art. In general, culturing is performed using an agar medium which is an LB medium frequently used for culturing Escherichia coli and to which an antibiotic according to a resistance gene in a vector is added.

  According to the present invention, silica fine particles can be obtained by culturing E. coli formed with the expression vector prepared as described above using a silicate-containing medium. Here, a liquid medium is used as the medium, and various kinds of medium conventionally used for culturing E. coli can be applied. For example, a 2 × YT medium is preferable as the medium, but the medium is not limited thereto. It is not something.

  Furthermore, a feature of the method for producing silica fine particles according to the present invention includes a step of culturing using a medium containing silicic acid (silicic acid). Means for this are roughly divided into two. The first means is to prepare silicic acid by previously mixing silanes and dilute hydrochloric acid, and add the mixed liquid (silicic acid) to the liquid medium. Here, silane means silicon hydride and its derivatives. For example, tetramethoxysilane is preferable as a silane to be used in the present invention, but is not limited thereto.

  The second means for making the medium contain silicic acid in the method of the present invention is to use the water in which the diatom is alive as the water for preparing the liquid medium. That is, a medium (for example, 2 × YT medium) containing an antibiotic is prepared using a supernatant of water in which diatoms are alive (for example, pond water).

The silica fine particles obtained by the method of the present invention are generally confirmed to be spherical particles in the range of 100 nm to 10 μm by observation with an electron microscope or the like.
Examples of the present invention will be described in more detail below, but the present invention is not limited to these examples.

Construction of Expression Vector An expression vector was constructed according to FIG.
10 μg each of two oligonucleotides synthesized with a DNA synthesizer (oligonucleotides 1 and 2 in FIG. 1) were mixed and heated to 90 ° C. The oligonucleotides were annealed (to bind complementary sequences) by further cooling to 50 ° C. over 40 minutes.
Next, a double-stranded DNA consisting of the base sequence of SEQ ID NO: 1 and its complementary sequence was synthesized by subjecting it to an extension reaction with DNA polymerase using a Blunting Kit [Takara Bio Inc.]. This double-stranded DNA was introduced into the EcoR V site of Bluescript II KS (+) [Toyobo Co., Ltd.], and the resulting plasmid (cloning vector) was designated as pBS-sil 1A.
After one amplification by a normal cloning method, the desired plasmid vector pBs-sil 1A was obtained by color selection.
Next, pBS-sil 1A was digested with restriction enzymes BamH I and Hind III [Takara Bio Inc.], and the resulting DNA fragment was recovered from the gel after agarose electrophoresis. The recovered DNA fragment is expressed as BamH of expression vector pET30c [Takara Bio Inc.]
Escherichia coli DH5α [Toyobo Co., Ltd.] was transformed with the plasmid obtained by binding to I and Hind III fragment.
The plasmid was purified from the transformed Escherichia coli by the alkaline method, and it was confirmed by electrophoresis after restriction enzyme treatment and PCR whether an expression vector was constructed. Specifically, by confirming a DNA fragment of about 100 bp, it was confirmed whether a desired expression vector was constructed, and the obtained expression vector was designated as pET30c-sil 1A.

Production of silica fine particles The expression vector pET30c-sil 1A prepared in Example 1 was used to transform E. coli BL21 (DE3) pLysS [Takara Bio Inc.] and LB containing antibiotics (kanamycin, chloramphenicol) Colonies that appeared on the agar medium were cultured in 10 ml of 2 × YT liquid medium containing antibiotics (kanamycin, chloramphenicol).
The culture broth overnight was transferred to 1 liter of 2 × YT medium. When the turbidity of the culture solution reached about 0.8, IPTG was added to a final concentration of 1 mM, and the culture was further continued for 3 and a half hours. When silicic acid prepared using tetramethoxysilane and dilute hydrochloric acid was added 15 minutes before the culture was stopped, silica spheres having a diameter of about 200 nm to 1 μm were formed as precipitates. The culture solution was centrifuged to collect the cells and silica spheres.
Alternatively, a 2XYT medium containing antibiotics (kanamycin, chloramphenicol) is used after centrifuging the water of a natural pond where diatoms live in the campus of the university where the inventor works. In the same manner as described above, E. coli BL21 (DE3) pLysS transformed with the expression vector pET30c-sil 1A was cultured (however, tetramethoxysilane and dilute hydrochloric acid were not added immediately before the culture was stopped). . As in the above case, formation of silica spheres of about 200 nm to 10 μm was observed.

  The present invention contributes to a simple and inexpensive manufacturing technology for silica fine particles (nanosilica), which is expected to be widely used as electrical / electronic materials, optical materials, enzyme-immobilized materials, various binders or fillers, etc. It can be used and developed in various fields.

It is a construction diagram of an expression vector used in the present invention.

Claims (4)

A step of culturing Escherichia coli transformed with an expression vector containing the following base sequence (SEQ ID NO: 1) and a complementary sequence thereof as a gene encoding a peptide to be expressed using a silicate-containing medium. A method for producing silica fine particles.

  2. The method for producing silica fine particles according to claim 1, wherein the silicic acid-containing medium is prepared by adding tetramethoxysilane and dilute hydrochloric acid.   A method for producing silica fine particles, characterized in that a silicic acid-containing medium is prepared using water in which diatoms are alive. E. coli transformed with an expression vector comprising the base sequence consisting of SEQ ID NO: 1 and its complementary sequence.

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