WO2019082935A1

WO2019082935A1 - Nucleotide construct for expressing spider silk protein in photosynthetic bacterium

Info

Publication number: WO2019082935A1
Application number: PCT/JP2018/039521
Authority: WO
Inventors: 圭司沼田; チューンピンフーン
Original assignee: 国立研究開発法人理化学研究所
Priority date: 2017-10-26
Filing date: 2018-10-24
Publication date: 2019-05-02
Also published as: JP2023116685A; JPWO2019082935A1

Abstract

The present inventor succeeded in introducing a nucleotide construct, said nucleotide construct containing a nucleotide encoding a spider silk protein and being functionally linked to a promoter capable of inducing protein expression in a photosynthetic bacterium, into the photosynthetic bacterium and expressing the protein. The photosynthetic bacterium can be maintained without feeding, which makes it possible to keep down the cost for the synthesis of the spider silk protein.

Description

Nucleotide constructs for expressing spider silk proteins in photosynthetic bacteria

The present invention relates to nucleotide constructs for expressing spider silk proteins in photosynthetic bacteria. The present invention also relates to a photosynthetic bacterium comprising the construct and a method of producing spider silk proteins using the photosynthetic bacterium.

Spiders produce seven different types of yarn fibers according to their purpose. Seven kinds of yarn fibers are extracted respectively from seven different glands (major gland (main tube foot capsule gland), feminine gland, squamous gland, grape gland, grape gland, piriform gland, collective gland, tubular gland) .

The main component of spider silk fiber is protein, and it has three highly conserved domains as its main structure. An alanine rich region (crystalline region, crystalline) and a glycine rich region (noncrystalline region, less-crystalline), with alternating core domains and unique amino-terminal and carboxyl-terminal domains on either side of it is there. And by having such a structure, it has a random coil structure, a beta sheet structure, and a helical structure, and the spider yarn fiber exhibits the outstanding characteristic by mixing of these structures.

For example, spider silk fibers are outstanding in material properties such as tensile strength and extensibility. Furthermore, it is also known that it is superior in toughness to iron and Kevlar (registered trademark), and spider silk fibers are the most tough materials in natural polymers and artificial polymers. Moreover, they are also useful in biomedical applications such as drug delivery and tissue engineering because they have biodegradability, biocompatibility and antimicrobial properties. Thus, spider silk fibers are attracting attention as promising supermaterials in a wide variety of fields.

However, large-scale production of spider silk proteins can only be achieved in heterologous hosts, as spiders cannibalize and turf, for example, expression of recombinant spider silk proteins can be achieved by bacteria, yeast (Pichia pastoris), Success has been reported in insects (silk Bombyx mori), plants (tobacco and potato) and animals (mouse and goat) (Non-patent Documents 1 and 2).

The object of the present invention is to make it possible to produce spider silk proteins at low synthesis costs.

As a result of intensive studies to achieve the above objects, the present inventors use photoautotrophic organisms as hosts for producing spider silk proteins instead of heterotrophic organisms such as yeast, insects, plants and animals. I imagined that. The organism can be maintained without the need for feeding by using carbon dioxide etc. obtained from the environment and light, so if this organism can be used for the production of spider silk proteins, it can be synthesized It is possible to reduce the cost.

Therefore, the present inventors have repeated the repetitive sequence (hereinafter also referred to as "monomer") in the great bottle-like longitudinal protein 1 (MaSp1) derived from Nephila clavipes in the marine red non-sulfur photosynthetic bacteria (Rhodovulum sulfidophilum) It tried to make it express. More specifically, first, a MaSp1 monomer protein containing one of the monomers, or a plasmid vector encoding a MaSp1 multimer protein containing a plurality of the monomers is cloned in E. coli, and the photosynthesis is performed. Bacteria were introduced by conjugal transfer.

As a result, expression of MaSp1 monomer protein in the photosynthetic bacteria could be detected by SDS-PAGE and Western blotting. Similarly, MaSp1 multimeric protein can also be detected by western blotting, and the present invention has been completed.

That is, the present invention relates to a nucleotide construct for expressing spider silk protein, a photosynthetic bacterium comprising the construct, and a method of producing spider silk protein using the photosynthetic bacterium, and more specifically provides the following.
<1> A nucleotide construct comprising a spider silk protein-encoding nucleotide operably linked to a promoter capable of inducing protein expression in photosynthetic bacteria.
<2> The nucleotide construct according to <1>, wherein the photosynthetic bacterium is a red photosynthetic bacterium.
<3> The nucleotide construct according to <1> or <2>, wherein the promoter is a Tac1 promoter.
<4> The nucleotide construct according to any one of <1> to <3>, wherein the spider silk protein is a MaSp1 protein.
<5> A photosynthetic bacterium into which the nucleotide construct according to any one of <1> to <4> has been introduced.
<6> A method for producing spider silk protein,
Culturing the photosynthetic bacteria described in <5> under light irradiation;
Recovering spider silk proteins from the culture of photosynthetic bacteria obtained by the culture.

According to the present invention, spider silk protein can be produced without feeding the host, so that the synthesis cost can be reduced.

It is a figure which shows the outline of the plasmid DNA for making a spider silk protein (MaSP1 protein) fuse with a His tag etc. and expressing it in photosynthetic bacteria by conjugal transfer between bacteria. It is the schematic which expands and shows a part (lower left part) of plasmid DNA shown in FIG. Analysis of protein solution of photosynthetic bacteria (induction of expression by IPTG: 1 to 4 days and non-induction) into which plasmid DNA encoding MaSP1 monomer protein fused with His tag etc. is introduced by SDS-PAGE Is a photograph of the gel showing the results. In the figure, “M” indicates a lane on which ECL low range rainbow molecular weight markers were migrated, and “FT1” to “FT5” indicate the flow-through 1 ̃ obtained after binding the protein solution to a HisTrap HP column. 5 shows lanes in which each of 5 was electrophoresed, and “HP” indicates a lane in which the above-mentioned protein solution was purified using a HisTrap HP column and concentrated. Also, the arrow indicates the size or position of MaSP1 monomeric protein fused to His tag etc. on SDS-PAGE. A plasmid DNA encoding MaSP1 monomer protein fused with a His tag etc. is introduced into photosynthetic bacteria and E. coli, and protein solutions of these bacteria are analyzed by Western blotting. The photograph of the PVDF membrane shows the results. is there. In the figure, “M” indicates a lane on which ECL low range rainbow molecular weight marker is migrated, and “HP” indicates a protein of photosynthetic bacteria (induction induction by IPTG: 1 to 4 days and non-induction) into which the plasmid DNA is introduced. The lysate was purified using a HisTrap HP column, and further concentrated was shown to indicate a migrating lane, and "1" represents a lane in which a protein lysis solution of photosynthetic bacteria (expression induction by IPTG: 3 days) into which the plasmid DNA had been introduced was migrated. “2” indicates a lane on which a protein solution of E. coli (expression induction by IPTG: 4 hours) into which the plasmid DNA has been introduced migrates, and “3” indicates an E. coli into which the plasmid DNA is introduced (expression induction by IPTG) Shows a lane in which the protein solution of 24 hours was run. Also, the arrow indicates the size or position of MaSP1 monomeric protein fused with His tag etc. on the PVDF membrane. A plasmid DNA encoding MaSP1 monomeric protein or MaSP1 multimeric protein fused with His tag etc. was introduced into photosynthetic bacteria and cultured for 4 days without induction of expression by IPTG. FIG. 5 is a photograph showing the results of analysis of the protein solution of the bacteria by SDS-PAGE (left in the figure) and Western blotting (right in the figure). In the figure, "1 M", "2 M", "3 M" and "6 M" are photosynthetic bacteria which expressed MaSP1 monomer protein, MaSP1 dimer protein, MaSP1 trimer protein and MaSP1 hexamer protein, respectively. Shows the result of analyzing.

(Nucleotide construct)
The nucleotide construct of the present invention is characterized in that it comprises a nucleotide encoding a spider silk protein functionally linked to a promoter capable of inducing protein expression in photosynthetic bacteria.

The "spider" in the present invention is an animal classified into arachnids, preferably an anthropomorphic spider, more preferably a spider, a spider, and more preferably a spider.

The “spider thread” or “spider thread” is also referred to as spider silk thread, and is produced by silk glands in the spider's body, and is spit from a spinneret of a spinneret (pinus) at the rear of the abdomen. The silk glands in the spider's body are divided into pyriform glands, vine-like glands, vitreous glands, tubular glands, tubular glands, collecting glands and mossy glands according to their shape. "A spider silk" is generally divided into pull yarns (alias: bookmark yarn, yarn), bow yarns, warp yarns, weft yarns, silk yarns, etc. from the function and components thereof.

The "arachnoid thread protein" encoded by the nucleotide construct of the present invention is a protein (analogous protein) having the characteristics of a protein constituting a spider thread, as well as a protein constituting a spider thread. The "arachnoid protein" according to the present invention does not have to have exactly the same sequence as a naturally occurring spider silk protein, but may be an artificially modified protein, but the "arachnid silk protein" according to the present invention It is preferable that the protein includes a structure in which a crystalline region and a glycine rich region (noncrystalline region) involved in the formation of a random coil structure are alternately arranged.

Examples of spider silk proteins typically include spidroin proteins. Spidroin protein is also referred to as fibroin, and is spun by a large spider gland of a natural spider and the like, and mainly includes spidroin I (MaSp1) and spidroin II (MaSp2). Examples of amino acid sequences of spidroin-related proteins and the like typically include the polypeptides described in the accession numbers shown in Tables 1 to 4 below, which are contained in the US National Center for Biotechnology Information (NCBI).

The sequence of a gene encoding a protein is naturally mutated, and the amino acid sequence of the protein may be changed accordingly. Therefore, the "spider proteins" according to the present invention include not only proteins specified in the typical sequences shown in Tables 1 to 4 (wild-type spider proteins) but also proteins consisting of naturally mutated sequences (wild-type) It is to be understood that homologs of type spider silk proteins, naturally occurring variants of wild type spider silk proteins, are also included.

In addition, “the spider silk protein” according to the present invention is not only such a naturally occurring protein (wild-type spider silk protein, a homolog of wild-type spider silk protein, and natural variants thereof) but also the above-mentioned. As such, the amino acid sequence may be artificially modified (a spider silk protein variant).

In addition, the "spider silk protein" may be not only full length but also a partial fragment thereof. The “partial fragment” is not particularly limited, and, for example, a repeating unit (monomer) including a structure in which an alanine rich region and a glycine rich region are alternately arranged, a core domain in which the repeating unit is repeated, A non-repetitive domain (non-repetitive amino terminal domain, non-repetitive carboxyl terminal domain) located at both ends of the core domain, preferably a MaSp1 monomer consisting of the amino acid sequence set forth in SEQ ID NO: 1 A protein is mentioned (In addition, the said monomer protein is derived from MaSp1 protein specified by accession number: P19837). In addition, as shown in Examples described later, a protein containing one of the monomer proteins (for example, a protein consisting of the amino acid sequence described in SEQ ID NO: 2) is suitably used as a spider silk protein. Furthermore, a MaSp1 multimeric protein containing a plurality of the above monomeric proteins is also suitably used. Examples of such multimeric proteins include a protein consisting of the amino acid sequence set forth in SEQ ID NO: 3 when two of the above monomeric proteins are contained, and, for example, when three of the above monomeric proteins are contained, A protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 is mentioned, and when it contains 6 of the above-mentioned monomeric proteins, for example, a protein consisting of the amino acid sequence set forth in SEQ ID NO: 5 is mentioned.

"Arachnoid protein" according to the present invention also includes a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the above-mentioned typical amino acid sequence. Here, "plural" is not particularly limited, but is usually 2 to 60, preferably 2 to 50, more preferably 2 to 40, still more preferably 2 to 30, more preferably 2 to 20. And more preferably 2 to 10 (eg, 2 to 8, 2 to 4, 2).

In addition, the “spider silk protein” according to the present invention preferably has 50% or more (eg, 60% or more, 70% or more) homology with the above-mentioned typical amino acid sequence, and 80% or more (eg, , 85% or more, 86% or more, 87% or more, 88% or more, 89% or more), more preferably 90% or more (eg, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more) is more preferable. Sequence homology can be determined using the BLASTP (amino acid level) program (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). The specific method of the analysis method using such a program is known and can be analyzed using default parameters. In addition, "homology" according to the present invention includes "identity" and "similarity".

In addition, "other proteins" may be added directly or indirectly to "the spider silk protein" encoded by the nucleotide construct according to the present invention. There is no particular limitation on "other proteins", and for the purpose of facilitating purification of spider silk protein according to the present invention, polyhistidine (His) tag (tag) protein, FLAG-tag protein (registered trademark), Tag for purification such as Sigma-Aldrich), S-tag, glutathione-S-transferase (GST), etc. is suitably used, and in the case of the purpose of facilitating detection of spider silk protein according to the present invention, GFP Tag proteins for detection such as fluorescent proteins such as E.coli and chemiluminescent proteins such as luciferase are preferably used. In addition, in order to separate these other proteins from spider silk proteins, sequences recognized by cleaving enzymes such as thrombin recognition sequences and enterokinase recognition sequences are disposed between spider silk proteins and other proteins. May be

The spider silk protein to which such other proteins have been added is not particularly limited, but is preferably a protein consisting of the amino acid sequence set forth in SEQ ID NO: 7 or 8 from the viewpoint of better expression efficiency in photosynthetic bacteria described later . In the protein consisting of the amino acid sequence set forth in SEQ ID NO: 7, a tag protein consisting of a His tag, a thrombin recognition sequence, an S tag and an enterokinase recognition sequence sequentially from N-terminal (amino acid sequence set forth in SEQ ID NO: 6 And a protein in which a MaSp1 monomeric protein consisting of the amino acid sequence of SEQ ID NO: 2 is arranged, and the amino acid sequence of SEQ ID NO: 8 is described in SEQ ID NO: 7 The amino acid sequence of (1) excludes the thrombin recognition sequence and the proline residue immediately after S-tag (for details, see Table 5 below).

In addition, as shown in Examples described later, as spider silk proteins to which other proteins have been added, the protein consisting of the amino acid sequence described in SEQ ID NO: 6 from the N-terminal, described in SEQ ID NO: 3, 4 or 5 A protein in which a MaSp1 multimeric protein consisting of the amino acid sequence of SEQ ID NO: 10 is arranged is also suitably used in the present invention.

The nucleotide construct of the present invention should at least include a "promoter" capable of inducing expression of the protein in photosynthetic bacteria by being operatively linked to the nucleotide encoding the above-mentioned spider silk protein. Such "promoter" is not particularly limited, and, for example, Tac1 promoter (typically, a promoter consisting of the nucleotide sequence of SEQ ID NO: 14), lac promoter (typically, SEQ ID NO: 15) And puf promoter (typically, a promoter consisting of the nucleotide sequence set forth in SEQ ID NO: 16), but from the viewpoint of better expression efficiency in photosynthetic bacteria, the Tac1 promoter is preferred. . The promoter may also be an inducible promoter, for example by temperature, pH, hormones, metabolites (eg lactose, mannitol and amino acids), light, osmotic potential (eg salt induction), heavy metals or antibiotics What is induced is mentioned.

The "promoter" according to the present invention also includes a protein consisting of a nucleotide sequence in which one or more nucleotides are substituted, deleted, added and / or inserted in the above-mentioned typical nucleotide sequence. Here, "plural" is not particularly limited, but generally 2 to 20, preferably 2 to 15, and more preferably 2 to 10 (for example, 2 to 9, 2 to 8, 2 to 7) The number is preferably 2 to 6, more preferably 2 to 5 (eg, 2 to 4, 2 to 3, 2).

In addition, the “promoter” according to the present invention preferably has 50% or more (eg, 60% or more, 70% or more) homology with the above-mentioned typical nucleotide sequence, and 80% or more (eg, 85 or more) % Or more, 86% or more, 87% or more, 88% or more, 89% or more), more preferably 90% or more (eg, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more) It is more preferable that the above be 96% or more, 97% or more, 98% or more, and 99% or more. Sequence homology can be determined using the BLASTN program. The specific method of the analysis method using such a program is known and can be analyzed using default parameters.

Furthermore, the nucleotide construct of the present invention may be, besides the promoter, other control sequences that contribute to expression (transcription and translation) in the photosynthetic bacteria of nucleotides encoding spider silk proteins, such as replication origin, terminator, A poly A addition signal, a poly linker, an enhancer, a silencer, a ribosome binding site and the like can be appropriately included. Generally, a nucleotide encoding spider silk protein is located downstream of the promoter, and a terminator is located downstream of the gene.

Furthermore, in addition to the above-mentioned sequences controlling expression, sequences may be included that induce expression. As a sequence for inducing such expression, there is mentioned a lactose operon which can induce the expression of a gene located downstream by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG).

In addition, when introduced into photosynthetic bacteria by conjugal transfer, the nucleotide construct of the present invention preferably contains at least one gene selected from the mob, tra, oriT and oriV gene groups. Although these genes are genes necessary for conjugal transfer between bacteria, they do not have to be all on the same nucleotide construct, and they are divided into another nucleotide construct (such as helper plasmid DNA) and conjugated by combining them. It can communicate.

Furthermore, the nucleotide construct of the present invention may contain a marker gene from the viewpoint that the transformed photosynthetic bacteria can be selected using the expression as an index. Examples of marker genes include kanamycin resistance gene, ampicillin resistance gene, chloramphenicol resistance gene, drug resistance gene such as hygromycin resistance gene, auxotrophic gene, luciferase gene, β-galactosidase gene, chloramphenicol acetyl gene Enzyme genes (reporter genes) such as transferase (CAT) gene can be mentioned.

In addition, as a form of such nucleotide construct, for example, cloning vectors such as plasmid DNA, cosmid DNA, phage DNA and the like can be mentioned, but it is not particularly limited thereto, but plasmid DNA is preferable, pBBR1MCS2, pKT230 And pBHR1 etc. are more preferred.

The procedures and methods for preparation of nucleotide constructs of the present invention can be those commonly used in the field of genetic engineering. For example, in order to prepare the nucleotide construct of the present invention, as shown in the examples below, the spider silk protein and, if necessary, nucleotides encoding other proteins are cleaved with an appropriate restriction enzyme, A method of insertion and ligation at a restriction enzyme site or multiple cloning site of a vector is employed.

In addition, it is preferable that the nucleotide coding for the spider silk protein etc. inserted in this way be optimized in accordance with the host cell for the frequency of use of codons in order to increase the expression amount in the host cell. Such codon optimization can be performed by known methods. The optimization also includes deletion of infrequently used codons (rare codons) as well as synonymous substitution for frequently used codons. Furthermore, in the present invention, a host cell to be subjected to the usage frequency of codons is usually a photosynthetic bacterium, but when using the conjugation transfer method described later, not only the recipient photosynthetic bacteria but also the donor photosynthetic bacteria. The frequency of use of codons may be optimized in accordance with the bacterium (E. coli in the following example).

(Transformant of photosynthetic bacteria)
The present invention provides photosynthetic bacteria into which the nucleotide construct described above has been introduced.

In the present invention, the “photosynthetic bacteria” may be any bacteria capable of photosynthesis, and may be non-oxygenated photosynthetic bacteria, or may be oxygenogenic photosynthetic bacteria (blue-colored bacteria). Non-oxygenated photosynthetic bacteria include red bacteria (red non-sulfur bacteria, red sulfur bacteria), green bacteria (green non-sulfur bacteria, green sulfur bacteria) and heliobacteria, but they can survive even under aerobic conditions. Because it is easy to handle and maintain, it is preferably a red-colored bacterium, and it can be grown under aerobic, dark or anaerobic light conditions, and it is easier to handle and maintain. In view of the fact that the culture is carried out under conditions of high salinity such as seawater, and also from the viewpoint that cross contamination during culture is easy to minimize, red non-sulfur is more preferable. It is a bacteria. More specifically, as preferable examples of the "photosynthetic bacteria" according to the present invention, Rhodovulum sulfidophilum, Rhodopseudomonas (Afifella) marina, Rhodovulum euryhalinum, Rhodovulum imhoffii, Rhodovulum tesquicola, Rhodovulium visapatnamense, Roseospiramarina tiv prosife It can be mentioned.

There is no particular limitation on the method for introducing a nucleotide construct into such a photosynthetic bacterium (transformation method), for example, a transfer method, an introduction method using a peptide, an electroporation method, a lithium acetate method, a calcium phosphate method, spheroplast Methods include lipofection methods, DEAE dextran methods, and methods using liposomes (cationic, fusogenic, pH sensitive, etc.).

In addition, whether or not photosynthetic bacteria have been transformed by such a method can be detected by detecting nucleotides encoding spider silk proteins by PCR, sequencing or the like, or detecting the proteins by an immunological technique (such as Western blotting) It can confirm by doing. Furthermore, when the nucleotide construct of the present invention contains the above-mentioned marker gene, it can also be confirmed by culturing under selection conditions corresponding to the marker.

(Method for producing spider silk protein)
The spider silk protein production method of the present invention is
Culturing the photosynthetic bacteria of the present invention under light irradiation;
And collecting the spider silk protein from the culture of photosynthetic bacteria obtained by the culture.

In the culture step according to the present invention, light to be irradiated to the photosynthetic bacteria may be any light having a wavelength that can be absorbed by the bacteria, for example, red light with a wavelength of 620 nm or more for red photosynthetic bacteria. 700 nm, for example 650 to 700 nm, specifically 680 nm, far-red light (wavelength: 700 to 800 nm, for example 700 to 750 nm, specifically 730 nm) are preferably used. Moreover, the light (for example, white light) which the light of the other wavelength mixed may be used. Furthermore, the irradiance is not particularly limited, and is usually 10 to 50 Wm ⁻² . Further, such light irradiation can be performed by using a light source such as a light emitting diode (LED), a laser light source, an artificial light source such as a fluorescent lamp, or a natural light source (sunlight).

There is no particular limitation on the culture medium used for culturing the photosynthetic bacteria, and it is sufficient if the conditions (organic substance, hydrogen donor such as hydrogen sulfide and water, carbon source) necessary for each photosynthesis are aligned, and photosynthetic bacteria are marine. In this case, a culture medium of seawater component (for example, marine agar, marine broth, seawater (sterile ocean water etc.) itself) is suitably used.

In addition, the method of the present invention can be cultured using an extremely inexpensive raw material such as an inorganic salt, as compared with an expression system etc. of E. coli. For example, instead of a carbon source such as glucose, fructose, gluconic acid and the like usually used in culture of E. coli, in the present invention, it is possible to culture using an inorganic carbon raw material such as carbon dioxide and carbonate. Furthermore, instead of organic nitrogen sources such as yeast extract and peptone, in the present invention, it is also possible to culture with inorganic nitrogen sources such as nitrogen, ammonia, ammonium salts, nitrates and nitrites.

As other conditions, it is preferable to culture under semi-aerobic (semi-anaerobic) conditions, under anaerobic conditions, and the culture temperature is usually 15 to 37 ° C., preferably 25 to 35 ° C. The culture time is not particularly limited, and may be appropriately adjusted depending on the nucleotide construct used, type of photosynthetic bacteria and introduction method, and degree of production of spider silk protein, but it is usually 1 to 30 days, preferably 2 It is preferably 10 days, more preferably 3 days.

Further, the “culture” of the photosynthetic bacteria thus obtained by culture is not only the photosynthetic bacteria grown in the present invention, but also the secretion products of the bacteria and the metabolism of the photosynthetic bacteria obtained by culturing. A culture medium containing products and the like, including dilutions and concentrates thereof.

There is no particular limitation on the recovery of spider silk protein from culture of such photosynthetic bacteria, and it can be carried out using known recovery and purification methods, for example, dissolving or mechanically destroying photosynthetic bacteria. Can be recovered by When spider silk proteins are secreted, they can be obtained by recovering the culture medium. The resulting protein can be purified using standard procedures. If desired, the harvest can be centrifuged to collect the appropriate fractions (precipitate or supernatant). Also, for further purification of spider silk proteins, it can be subjected to gel filtration chromatography such as anion exchange chromatography, dialysis, phase separation or filtration. Furthermore, when a tag protein for purification is added to a spider silk protein and added to a photosynthetic bacterium, it can be purified using affinity chromatography according to the tag.

In the method of the present invention, as described above, it is possible to culture only with the inorganic salt raw material free from contaminating proteins. Thus, rather than other host cells such as E. coli, the method of the invention can simplify the cumbersome steps in protein purification.

Hereinafter, the present invention will be more specifically described based on examples, but the present invention is not limited to the following examples. In the present example, expression of spider silk protein in photosynthetic bacteria was attempted using the following materials and methods.

The spider silk protein which was attempted to be expressed in the present example is a protein containing a repetitive sequence (monomer) in MaSp1 of a spider (Nephila clavipe), its dimer, trimer and hexamer respectively ( Hereinafter, these proteins are also collectively referred to as "MaSp1 protein". In addition, a tag protein consisting of a His tag, a thrombin recognition sequence, an S tag and an enterokinase recognition sequence (a protein consisting of the amino acid sequence described in SEQ ID NO: 6) is fused to the N terminal of these MaSp1 proteins, Expression was tried (hereinafter, the protein in which the tag protein is fused is also generically referred to as "fused MaSp1 protein"). Table 6 below shows the amino acid sequence (SEQ ID NO :) of each MaSp1 protein, and the number of amino acids and the molecular weight of each fused MaSp1 protein.

(Bacterial strain and culture conditions)
The marine red non-sulfur photosynthetic bacterium Rhodovulum sulfidophilum DSM 1374 / ATCC 35886 was obtained from RIKEN Microorganism Materials Development Laboratory (JCM). Rdv. The sulfidophilum was cultured (maintained) in marine agar or phosphorus broth (BD Difco, USA) at 30 ° C. under semi-aerobic conditions under continuous irradiation of far-red light (730 nm, 30 Wm ⁻² ).

Maintain Escherichia coli DH5α (TaKaRa, Japan) in shaking culture at 180 ° C. under aerobic conditions at 37 ° C. in Lysogeny broth (LB) agar or LB broth (BD Difco LB, USA) It served for general cloning.

Rdv. In order to perform plasmid junctional transfer to S. sulfidophilum, coli S17-1 (Simon, R. et al., Nature Biotechnology, 1983, 1 (9): 784-791.) is used as a donor strain in LB agar or LB broth (BD Difco, USA), The culture was maintained at 37 ° C. under aerobic conditions and shaking culture at 180 rpm.

(Plasmid construction and conjugal transfer to Rdv. Sulfidophilum)
All PCR amplification was performed using KOD-plus DNA polymerase (TOYOBO, Japan). In addition, each of the above-described MaSp1 proteins encodes E. coli. pET30-a-MaSp1 (Numata, K), together with a DNA encoding a Co. coli-codon-optimized DNA, together with a DNA encoding His tag, S tag, thrombin recognition sequence and enterokinase recognition sequence (amino acid sequence described in SEQ ID NO: 6). Et al., Advanced Drug Delivery Reviews, 2010, 62 (15): 1497-1508), using the primers shown in Table 7 below (SEQ ID NOS: 11-13). Further, in the table, the Tac1 promoter is also shown together with the primers (SEQ ID NOS: 9 and 10) used to amplify the plasmid having the promoter as a template.

In Table 7, underlined portions indicate restriction enzyme recognition sequences. Also, the portions described in bold indicate ribosome binding sequences (RBS).

The TacI promoter and MaSp1 gene sequences are treated with the corresponding restriction enzymes and purified, and then the host vector pBBR1MCS-2 (Kovach, M. E. et al., Gene, 1995, 166 (1): 175-176). (See FIGS. 1 and 2).

E. carrying a recombinant plasmid coli S17-1 (donor) and Rdv. Bacterial junctional transfer with sulfidophilum (recipient) was performed according to the method described below.

First, E. coli carrying the recombinant plasmid. E. coli S17-1 was inoculated into 5 mL LB medium containing 50 μg mL ⁻¹ kanamycin, and shake culture was performed at 37 ° C. and 180 rpm for 16 hours. Meanwhile, Rdv. The sulfidophilum was inoculated in 15 mL of marine broth and cultured at 30 ° C. under semiaerobic conditions under continuous irradiation of far-red light (730 nm, 30 Wm ⁻² ) for 30 hours.

Both bacterial cultures were subjected to centrifugation at 12,000 rpm for 3 minutes and then resuspended in fresh medium (E. coli S17-1 in LB broth and Rdv. Sulfidophilum in marine broth). did).

And E. coli S17-1 and Rdv. The cell suspension of sulfidophilum was mixed at 1: 1. About 200 μL of the obtained bacterial mixture was soaked in a marine agar plate and cultured for 1 day under continuous irradiation of far-red light (730 nm, 30 Wm ⁻² ). The cells were then scraped from the agar and resuspended in 5 mL of fresh marine broth.

The resulting cell suspension to about 100 [mu] L, plated on 100MyugmL ^-1 kanamycin and 100MyugmL ^-1 potassium tellurite containing Simmons citrate agar. The agar plates were cultured at 30 ° C. for 7 days under continuous irradiation of far-red light (730 nm, 30 Wm ⁻² ).

In addition, it was confirmed by colony PCR, restriction enzyme treatment, and DNA sequencing that the conjugation completion body was obtained.

(Expression of fusion MaSp1 protein)
E. recombinant E. coli into which the pET30 plasmid containing the MaSp1 gene from Nephila clavipes optimized for the E. coli codon was introduced. E. coli BL21 (DE3) (TaKaRa, Japan) at 37 ° C. in 50 μg mL ⁻¹ kanamycin containing LB broth while shaking (180 rpm) to OD ₆₀₀ (the turbidity of the culture solution) to about 1.0 It was cultured to reach the end. Thereafter, the expression of MaSp1 protein was induced by adding 1 mM isopropyl-β-thiogalactopyranoside (IPTG) and changing the culture temperature to 30 ° C. and culturing for 4 hours or 24 hours.

E. recombinant Rdv. transduced with MaSp1 gene from Nephila clavipes optimized for codons of E. coli. C. sulfidophilum was cultured at 30 ° C. in 100 μg mL ⁻¹ kanamycin-containing marine broth under semiaerobic conditions, continuous irradiation with far-red light (730 nm, 30 Wm ⁻² ) until the OD ₆₀₀ reached about 1.5. did. Then, expression induction of the fusion MaSp1 protein was performed by adding 1 mM IPTG and culturing it for 1, 2, 3 and 4 days. Also, after the OD ₆₀₀ reached about 1.5, the cells were further cultured for 4 days in the absence of IPTG.

(Preparation of protein solution)
After IPTG induction or culture under non-IPTG induction, bacteria were recovered by centrifugation at 9,000 g for 10 minutes at 4 ° C. Bacterial pellets at a wet weight of 1 g were resuspended in 5 mL of denaturation buffer (10 mM Tris, 8 M urea and 100 mM NaH ₂ PO ₄ , pH 7). Furthermore, the cell suspension was stirred overnight at 4 ° C. and subjected to centrifugation at 9,000 g for 30 minutes at 4 ° C. Then, the supernatant (protein solution) was collected.

(HisTrap purification and concentration)
Combine protein lysates (cultured under IPTG induction or not for 1 to 4 days) (approx. 550 mL culture solution) and use HisTrap HP 1 mL column (GE Healthcare Life Sciences, USA) Purified according to the manufacturer's protocol.

In purification, binding buffer (8 M urea, 0.5 M NaCl, 20 mM phosphate buffer, 5 mM imidazole, pH 7.4) and extraction buffer (8 M urea, 0.5 M NaCl, 20 mM phosphate buffer, 500 mM imidazole, pH 7.4) Was filtered through a 0.22 .mu.m cellulose acetate filter (Corning, USA).

The purified product obtained was concentrated using a Vivaspin 6 MWCO 3000 protein-concentrated spin column (GE Healthcare Life Sciences, USA) using a HisTrap HP 1 mL column, a binding buffer and an extraction buffer.

Since the fused MaSp1 protein in which the 6 × histidine residue (His tag) is fused to the N-terminus has the ability to bind to the HisTrap column, it is purified and concentrated by the above treatment.

(SDS polyacrylamide gel electrophoresis (SDS-PAGE))
The sample prepared above (protein solution, protein solution after concentration with HisTrap, etc.) was subjected to SDS-PAGE using 16.5% pre-cast Tris-Tricine gel (Bio-Rad, USA) for 2 hours. . Then, the gel was impregnated with fixing buffer (25% ethanol and 15% formaldehyde) for 30 minutes and then subjected to staining with Coomassie Brilliant Blue (CBB) -R250 for 1 hour. The obtained result is shown in FIG.

(Western blotting)
Using the semi-dry blotter (Bio-Rad, USA) to the polyvinylidene fluoride (PVDF) membrane (0.2 μm pore size) (Bio-Rad, USA) from the gel subjected to the SDS-PAGE, using a semi-dry blotter (Bio-Rad, USA) Were electrophoretically transferred.

Detection of fused MaSp1 monomeric protein containing His tag in a PVDF membrane is carried out according to His tag (registered trademark) Western reagent protocol (Novagen, USA) according to anti-His tag monoclonal antibody and AP-labeled goat anti-mouse IgG Using a His-tag AP Western reagent. The obtained result is shown in FIG. In addition, detection of the fusion MaSp1 protein can be carried out according to the above-mentioned protocol: anti-His tag monoclonal antibody and HRP-labeled goat anti-mouse IgG (Thermo Fisher Scientific, USA), and Novex (registered trademark) ECL chemiluminescence substrate reagent kit ( Chemiluminescence detection was also performed using Thermo Fisher Scientific (USA). The obtained result is shown in FIG.

(LC-MS / MS analysis)
The target band was excised from the gel subjected to the SDS-PAGE and analyzed by liquid chromatography / mass spectrometry (LC-MS) to identify the amino acid sequence. The LC-MS data was processed and searched by the MASCOT program.

As is clear from the results shown in FIG. 3, the marine red non-sulfur photosynthetic bacteria Rdv. As a result of attempting to introduce into sulfidophilum, a strong band was detected at the position corresponding to the molecular weight of the protein in SDS-PAGE (see lane HP in FIG. 3). Furthermore, as apparent from the results shown in FIG. 4, in Western blotting using an anti-His tag antibody, a His tag fused to the MaSp1 monomer protein was detected at the same position (see lane HP in FIG. 4). ). Further, as is clear from the results shown in FIG. 5, not only the MaSp1 monomeric protein but also any of dimeric protein, trimeric protein and hexameric protein could be detected. The fused MaSp1 monomeric protein was confirmed by LC-MS / MS analysis that the detected protein was the monomeric protein.

Therefore, it became clear that it is possible to express MaSp1 protein (spider silk protein) in photosynthetic bacteria.

As described above, according to the present invention, photosynthetic bacteria, which are sites for producing spider silk proteins, can be maintained and propagated without the need for feeding, so that the synthesis cost can be reduced. It becomes possible.

As mentioned above, spider silk proteins stand out in material properties such as tensile strength, extensibility and toughness. Therefore, it can be used, for example, in the manufacture and development of materials that require high impact resistance, such as bulletproof clothing, parachute, car bodies of automobiles, and the like. Furthermore, since they have biodegradability, biocompatibility and antibacterial properties, spider silk proteins are also used in the manufacture and development of medical materials such as wound closures, sutures, bandages and scaffolds for regenerative medicine. Available.

Therefore, the present invention which can provide such spider silk protein at low cost for its synthesis is extremely useful in various fields such as industrial fields and medical fields.

SEQ ID NOs: 2 to 8
<223> SEQ ID NO: 9 to 13 of artificially synthesized polypeptide
<223> SEQ ID NOs: 14 to 16 of artificially synthesized primers
<223> Sequence of artificially synthesized promoter

Claims

A nucleotide construct comprising a nucleotide encoding a spider silk protein operably linked to a promoter capable of inducing protein expression in photosynthetic bacteria.
The nucleotide construct according to claim 1, wherein the photosynthetic bacteria are purple photosynthetic bacteria.
The nucleotide construct according to claim 1 or 2, wherein the promoter is a Tac1 promoter.
The nucleotide construct according to any one of the preceding claims, wherein the spider silk protein is a MaSp1 protein.
A photosynthetic bacterium into which the nucleotide construct according to any one of claims 1 to 4 has been introduced.
A method of producing spider silk protein comprising
Culturing the photosynthetic bacteria according to claim 5 under light irradiation;
Recovering spider silk proteins from the culture of photosynthetic bacteria obtained by the culture.