JP6906215B2 - Vector for Streptomyces microorganisms - Google Patents

Description

The present invention relates to vectors for Streptomyces microorganisms.

Bacteria of the genus Streptomyces are widely used industrially as a group of actinomycetes that produce various secondary metabolites such as antibiotics, and are an extremely important group of bacteria. Considering the importance of producing useful substances in Streptomyces bacteria, it is desired to develop a large-scale expression system in Streptomyces bacteria.

In view of the importance of gene recombination technology for Streptomyces bacteria, the present inventors have a very strong application of the expression mechanism of high molecular weight nitrile hydratase (H-NHase) possessed by actinomycetes. We are developing a constitutive expression vector pHSA81, which is an expression vector (Patent Document 1).

Japanese Unexamined Patent Publication No. 2007-053994

In recent years, many secretory enzymes are used for industrial applications, so the development of a secretory expression vector is required as a method for more efficiently producing useful secretory enzymes. Among them, the purpose is to use the Tat secretory pathway. Vectors have been developed that enable mass secretory production of proteins.
This Tat secretory pathway has also been functionally analyzed in the genus Streptomyces, but its usefulness is extremely limited due to the inability to strictly control its expression.
On the other hand, the vector pHSA81 developed by the present inventors is useful as an experimental tool for heterologous expression, but the heterologous protein is expressed only intracellularly, and further, pHSA81 replicates only in Streptomyces actinomycetes. There was a problem that it was not possible and it took time to construct the plasmid.

Therefore, one of the objects of the present invention is to provide a constitutive secretion expression vector capable of realizing mass secretory production of a target protein by using a Tat secretory pathway that functions in Streptomyces bacteria. There is.
Another object of the present invention is to modify the vector pHSA81 into a constitutive expression shuttle vector that can be replicated by both Escherichia microorganisms and Streptomyces microorganisms.
Furthermore, by using the Tat secretory pathway that functions in Streptomyces bacteria, mass secretory production of the target protein can be achieved, and it can be replicated by both Escherichia microorganisms and Streptomyces microorganisms. One of the purposes is to provide a constitutive secretion expression shuttle vector.

That is, the present invention
(A) DNA region capable of expressing the target protein during culture using a Streptomyces microorganism as a host (b) DNA region capable of secreting and expressing the target protein during culture using a Streptomyces microorganism as a host (C) A constitutive secretion expression vector containing a DNA region capable of replicating using a microorganism of the genus Streptomyces as a host.

In addition, the present invention
(D) DNA region capable of expressing the target protein without adding an inducer during culturing using a Streptomyces microorganism as a host (e) DNA region capable of replicating using a Streptomyces microorganism as a host (f) Escherichia microorganism It is a constitutive expression shuttle vector containing a DNA region capable of replicating using the above as a host.

Furthermore, the present invention
(G) DNA region capable of expressing the target protein during culture using a Streptomyces microorganism as a host (h) DNA region capable of secreting and expressing the target protein during culture using a Streptomyces microorganism as a host (I) A DNA region capable of replicating using a microorganism of the genus Streptomyces as a host (j) A constitutive secretion expression shuttle vector containing a DNA region capable of replicating a microorganism of the genus Escherichia as a host.

Further, the present invention relates to a constitutive expression vector containing the regions (a) to (c), a constitutive expression shuttle vector containing the regions (d) to (f), or the above (g) to ( A transformant transformed from a Streptomyces microorganism by a constitutive secretion expression shuttle vector containing the region of j) is also provided.

Furthermore, the present invention also provides a method for producing a target protein, which comprises culturing the transformant and collecting the target protein from the obtained culture. Here, the target protein is, for example, a high molecular weight nitrile hydratase.

That is, the step of amplifying the DNA fragment using the DNA region encoding the target protein as a template.
A step of linking the amplified DNA fragment to a restriction enzyme-treated constitutive secretion expression vector.
A step of transforming a microorganism of the genus Streptomyces with a vector into which the DNA fragment has been introduced, and a step of culturing the Escherichia coli and collecting a target protein from the obtained culture supernatant.
A method for producing a target protein can be provided.

Further, a step of amplifying the DNA fragment using the DNA region encoding the target protein as a template.
A step of linking the amplified DNA fragment to a restriction enzyme-treated constitutive expression shuttle vector.
A step of transforming a microorganism of the genus Streptomyces with a vector into which the DNA fragment has been introduced, and a step of culturing the Escherichia coli and collecting a target protein from the obtained culture supernatant.
A method for producing a target protein can be provided.

Furthermore, the step of amplifying the DNA fragment using the DNA region encoding the target protein as a template.
A step of linking the amplified DNA fragment to a restriction enzyme-treated constitutive secretion expression shuttle vector.
A step of transforming a microorganism of the genus Streptomyces with a vector into which the DNA fragment has been introduced, and a step of culturing the Escherichia coli and collecting a target protein from the obtained culture supernatant.
A method for producing a target protein can be provided.

According to the present invention, mass secretory production of a target protein can be realized by using a Tat secretory pathway that functions in Streptomyces bacteria.
Further, according to the present invention, the construction can be carried out by a microorganism of the genus Escherichia having a short culture time, and the target protein can be expressed using the microorganism of the genus Streptomyces as a host.

It is a figure which shows the structural example of the constitutive secretion expression vector pHSA81-ss1053. FIG. 5 is a drawing relating to the result of electrophoresis showing that the msgLAP gene product is not secreted extracellularly. It is a graph substitute drawing which shows the LAP activity of the signal peptide provided by the constitutive secretion expression vector which concerns on this invention. It is a figure which shows the result of electrophoresis about the secretory ability of msgLAP of a signal peptide. It is a figure which shows the functional analysis result of the constitutive secretion expression vector which concerns on this invention. It is a figure which shows the functional analysis result in the plate assay of the constitutive secretion expression vector which concerns on this invention. It is a figure which shows the composition of the constitutive expression shuttle vector derived from pHSA81. It is a figure which shows the construction of the constitutive secretion expression shuttle vector pEHSA81k-ss1053 which concerns on this invention. It is a figure which shows the functional analysis result of the constitutive secretion expression shuttle vector which concerns on this invention. It is a figure which shows the functional analysis result in the plate assay of the constitutive secretion expression shuttle vector which concerns on this invention.

1. 1. Constitutive secretory expression vector according to the present invention The constitutive secretory expression vector according to the present invention is an inducer during culturing using a microorganism belonging to the genus Streptomyces (hereinafter, also referred to as “microorganism of the genus Streptomyces”) as a host. A DNA region capable of expressing the target protein without adding the substance, a DNA region capable of secreting and expressing the target protein during culturing using a Streptomyces microorganism as a host, and a DNA region capable of replicating using a Streptomyces microorganism as a host. ,including. That is, the constitutive secretion expression vector according to the present invention is a vector that can be replicated in a microorganism of the genus Streptomyces and is effective for secreting and expressing the target protein outside the microorganism of the genus Streptomyces.

(1) DNA region capable of expressing a target protein without adding an inducer during culturing using a Streptomyces microorganism as a host The constitutive secretion expression vector of the present invention is an inducer during culturing using a Streptomyces microorganism as a host. By introducing a DNA region capable of expressing the target protein into a host microorganism of the genus Streptomyces without adding the target protein, the target protein can be produced with extremely high efficiency without adding an expression inducer. Can be done.
More specifically, (i) the promoter of the H-nitrile hydratase (H-NHase) gene of Rhodococcus rhodochrous, and (ii) the H-nitrile hydratase transcriptional regulatory protein (hereinafter also referred to as "regulatory protein"). ) (NhhC) encoding gene, (iii) transcription activation protein (NhhD) encoding gene, (iv) multicloning site (MCS), and (v) terminator, utilizing H-NHase expression regulation mechanism Is what you do. The base sequence of the gene responsible for the expression regulation mechanism is described in JP-A-2007-053994 disclosed by the present inventors.

(I) Rhodococcus rhodochrous H-nitrile hydratase gene promoter In the present invention, the H-NHase gene promoter can be cut out from a cell carrying the H-NHase gene using a restriction enzyme. .. Alternatively, it can be obtained by designing a primer provided with an appropriate restriction enzyme recognition site and amplifying it by PCR using a desired promoter region in the upstream region of the structural gene of H-NHase as a template. It is also possible to chemically synthesize and use a desired promoter region based on the base sequence information of the region including the H-NHase gene promoter region that has already been known (for example, GenBank Accession number: D67027). The sequence and composition of the promoter of the H-NHase gene used in the vector of the present invention are described in JP-A-2007-053994.

(Ii) Gene encoding the regulatory protein (NhhC) The gene encoding the regulatory protein (NhhC) in the present invention can be excised from a cell carrying the nhhC gene using a restriction enzyme. Alternatively, it can be obtained by amplifying a desired nhhC gene region by PCR using a primer provided with a restriction enzyme recognition site. It is also possible to chemically synthesize a desired nhhC gene based on the already known base sequence information of the nhhC gene. The sequence and composition of the gene encoding this regulatory protein (NhhC) are described in JP-A-2007-053994.

(Iii) Gene encoding transcriptional activation protein (NhhD) The gene encoding transcriptional activation protein (NhhD) in the present invention can be excised from a cell carrying the NhhD gene using a restriction enzyme. Alternatively, it can be obtained by amplifying a desired NhhD gene region by PCR using a primer provided with a restriction enzyme recognition site. It is also possible to chemically synthesize a desired NhhD gene based on the already known base sequence information of the NhhD gene. The sequence and composition of the gene encoding this transcription activation protein (NhhD) are described in JP-A-2007-053994.

(Iv) Multi-cloning site (MCS)
Although the MCS in the present invention is not particularly limited, for example, one containing all six restriction enzyme sites of NdeI, BamHI, HindIII, NheI, SpeI, and XbaI is preferable. The sequence of MCS in the present invention is described in JP-A-2007-053994.

(V) Terminator The vector of the present invention requires a terminator region, and it is preferable that the terminator region is present downstream of the MCS of (iv) above.
This presence prevents the strong transcriptional activity from the H-NHase gene promoter from adversely affecting other regions downstream of the MCS (eg, replication region) and stabilizes the vector. Can be done. It is considered that this stabilizing effect greatly contributes to the improvement of the expression efficiency of the gene high expression system referred to in the present invention.

The terminator in the present invention can be cut out from cells, phages, or the like having a desired terminator region using a restriction enzyme. Alternatively, it can be obtained by amplifying a desired terminator region by PCR using a primer provided with a restriction enzyme recognition site. It is also possible to chemically synthesize a desired terminator region based on the already known base sequence information of the terminator region.

Further, as the terminator that can be used in the present invention, a terminator derived from any gene may be used as long as it has activity in the host, and the terminator derived from fd phage (fd-ter) may be used without limitation. ), Terminator derived from T4 phage (T4-ter), terminator derived from T7 phage (T7-ter) and the like are preferably mentioned. Of these, a terminator derived from fd phage is particularly preferable because it has a greater stabilizing effect as described above.

(2) DNA region capable of secreting and expressing a target protein during culture using a Streptomyces microorganism as a host The constitutive secretion expression vector according to the present invention can secrete and express a target protein during culture using a Streptomyces microorganism as a host. It contains a DNA region and can secrete and express the target protein outside the Streptomyces microorganism during culturing. Here, in order to realize the secretory expression of the protein, the vector of the present invention contains a predetermined signal peptide.

Although the signal peptide is not particularly limited, it is preferable to use a signal peptide selected by screening using a predetermined reporter protein. As this screening source, it is preferable to use Streptomyces avermitilis K139, which is a microorganism of the genus Streptomyces, whose entire genome sequence is determined and whose total secretory protein can be predicted. Furthermore, from the total secretory proteins from S. avermitilis K139, those predicted to depend on the Tat secretion pathway were selected, and the signal peptide prediction server SignalP4.0 (http://www.cbs.dtu.dk/) It is preferable that the selection is based on the D value calculated by services / SignalP /). The type of this signal peptide will be described later.

(3) DNA region that can be replicated using a microorganism of the genus Streptomyces as a host Since the constitutive secretion expression vector of the present invention contains a DNA region that can be replicated within the microorganism of the genus Streptomyces, it is within the microorganism belonging to the genus Streptomyces. Can be duplicated with. In order for the plasmid to be replicable within the Streptomyces microorganism, it suffices to contain a replication region (ori, rep) derived from the plasmid of the Streptomyces microorganism.

Further, in the constitutive secretion expression vector of the present invention, the DNA region capable of replicating using a Streptomyces microorganism as a host may further contain an antibiotic resistance gene for a Streptomyces microorganism in addition to the replication region. .. As the antibiotic resistance gene for Streptomyces microorganisms, a thiostreptone resistance gene or the like can be selected.

(4) Construction of Constitutive Secretion Expression Vector The constitutive secretion expression vector according to the present invention is constructed by introducing the signal peptide upstream of the MCS of the vector pHSA81 (Patent Document 1). FIG. 1 shows a configuration example of pHSA81-ss1053, which is one of the constitutive secretion expression vectors according to the present invention.

More specifically, first, the genomic DNA of the S. avermitilis K139 strain is used as a template, and the signal sequence fragment is amplified by a PCR reaction. Then, by a recombination reaction using GENEART, the amplified DNA fragment is ligated to the vector pHSA81 treated with a restriction enzyme (NdeI).
Furthermore, Streptomyces lividans TK24 is transformed by the PEG method using each GENEART reaction solution to obtain colonies. Then, the obtained colonies are picked and subcultured in YEME / tsr solid medium. The cells sufficiently grown on the subcultured YEME / tsr solid medium are inoculated into a 10 ml YEME / tsr liquid medium, and shake-cultured at 28 ° C. and 130 rpm for 4 days. Bacterial cells are collected from the culture solution by centrifugation, and a plasmid, which is a constitutive secretion expression vector, is extracted.
DNA sequence analysis is performed on this extracted plasmid to confirm that the target fragment is introduced upstream of the extracted plasmid while leaving the Nde I recognition site and that no mutation is contained. From this, it can be confirmed that the constitutive secretion expression vector is constructed.

(5) Transformant The transformant of the present invention can be prepared by transforming a host with a constitutive secretion expression vector of the present invention or a plasmid in which a target protein gene is introduced into the constitutive secretion expression vector. For the transformation, a conventional method may be used, for example, an electroporation method, a calcium phosphate method, or a DEAE dextran method can be used. The host used in the present invention is a microorganism belonging to the genus Streptomyces.

(6) Method for Producing Protein The present invention can also provide a method for producing a target protein. That is, the target protein can be produced by culturing the transformant of (5) above and collecting the target protein from the obtained culture. As the method for culturing the transformant, a method suitable for the microorganism of the genus Streptomyces used as the host may be appropriately selected.

Further, in the method for producing a protein according to the present invention, the "culture" means a product obtained by culturing a culture solution, a culture supernatant, cells, a cell-free extract, a cell membrane, or the like. In the cell-free extract, the cultured cells are physically crushed with a homogenizer or the like by adding, for example, sodium phosphate buffer, and then centrifuged (for example, 15,000 rpm, 10 min, 4 ° C.) to crush the cells that cannot be crushed. The supernatant can be collected and obtained so that (cells) are not present. The cell membrane can be obtained by suspending the pellet obtained by centrifugation in a lysis buffer.

As the target protein, the culture may be used as it is, or a known method such as dialysis or sulfur precipitation, electrophoresis, or various chromatography such as gel filtration, ion exchange, affinity, etc. may be used alone or in combination as appropriate. Therefore, concentrated and purified products may be used.

2. Constitutive expression shuttle vector according to the present invention The constitutive expression shuttle vector according to the present invention has a DNA region capable of expressing a target protein without adding an inducer during culturing using a microorganism of the genus Streptomyces as a host, and streptomyces. It includes a DNA region capable of replicating using a genus microorganism as a host and a DNA region capable of replicating a microorganism belonging to the genus Escherichia (hereinafter, also referred to as “Escherichia genus microorganism”) as a host. That is, the constitutive expression shuttle vector according to the present invention is a constitutive expression shuttle vector plasmid that can replicate in both Streptomyces microorganisms and Escherichia microorganisms. Each DNA region will be described below. Here, the above-mentioned present invention relates to a DNA region capable of expressing a target protein without adding an inducer during culturing using a Streptomyces microorganism as a host, and a DNA region capable of replicating using a Streptomyces microorganism as a host. Since it is the same as that provided by the constitutive secretion expression vector according to the above, description thereof will be omitted here.

(1) DNA region capable of replicating using a microorganism belonging to the genus Escherichia Since the vector of the present invention contains a DNA region capable of replicating within a microorganism belonging to the genus Escherichia, it can be replicated within a microorganism belonging to the genus Escherichia. In order for the plasmid to be replicable within the Escherichia microorganism, it may contain a replication region (ColEI) derived from the plasmid of the Escherichia microorganism. As a DNA region that can be replicated in a microorganism of the genus Escherichia, plasmids such as pBluescriptII SK (-) (Agilent Technologies Co., Ltd.), pHSG398, pHSG298 (Takara Bio Inc.) can be used and have a replication region. As long as it is, it may be the whole plasmid or a part of the plasmid.

Further, in the constitutive expression shuttle vector of the present invention, the DNA region capable of replicating using an Escherichia microorganism as a host may further contain an antibiotic resistance gene for an Escherichia microorganism in addition to the replication region. As the antibiotic resistance gene for microorganisms of the genus Escherichia, a chloramphenicol resistance gene, a kanamycin resistance gene, an ampicillin resistance gene and the like can be selected.

Here, in the present invention, as the Escherichia microorganism, Escherichia coli is preferable, and more specifically, TOP10, DH10B, Stbl2, Stbl3, Stbl4 (all Invitrogen) and the like are mentioned, and among these, Escherichia coli is mentioned. , TOP10 and Stbl2 are more preferred.

(2) Construction of Constitutive Expression Shuttle Vector An example of a method for constructing a constitutive expression shuttle vector according to the present invention will be described.
First, a DNA fragment containing the origin of plasmid replication of Escherichia coli and the antibiotic resistance gene for Escherichia microorganisms is amplified by PCR using the expression vector pBluescriptII as a template.
Next, the amplified DNA fragment is ligated to the vector pHSA81 treated with a restriction enzyme (for example, BlpI) by a recombination reaction using GENEART. Furthermore, Escherichia coli TOP10 and Escherichia coli stbl2 are transformed by the heat shock method using each GENEART reaction solution to obtain colonies.
The obtained colonies are picked and subcultured in 2YT solid medium containing each drug. Furthermore, each cell grown on a 2YT solid medium containing each drug was inoculated into a 2YT liquid medium containing 5 ml of each drug, and the medium became sufficiently turbid at 37 ° C. and 150 rpm (at 37 ° C. and 150 rpm). Cultivate for about 12 hours). The plasmid is extracted from the cultured cells to obtain a constitutive expression shuttle vector.

(3) Stability test of plasmid In the constitutive expression shuttle vector constructed in this manner, it is necessary to test the stability of the plasmid in order to confirm the shuttle formation. The stability test of the constitutive expression shuttle vector according to the present invention can be carried out, for example, as follows.
Inoculate 1% of the pre-liquid culture medium into a new 5 ml 2YT liquid medium containing each drug, and incubate again for 12 hours. This is subcultured again, and after the third subculture, the cells are collected and the plasmid is extracted.
On the other hand, S. lividans TK24 is transformed with each plasmid by the PEG method to obtain colonies.
Each cell sufficiently grown on the YEME / tsr solid medium is inoculated into a 10 ml YEME / tsr liquid medium in 2 mm squares, and shake-cultured at 28 ° C. and 130 rpm for 4 days. Next, the culture solution is subcultured in a new 10 ml YEME / tsr liquid medium culture solution with 1% inoculation, and the plasmid is extracted from the cells that have undergone a total of 3 subcultures.
After 3 subcultures with Escherichia coli and 3 subcultures with actinomycetes, each plasmid is treated with a restriction enzyme (for example, BlpI) and subjected to agarose gel electrophoresis. Then, the stability of the plasmid is examined from the band pattern of the DNA fragment.

By extracting the plasmid under the above conditions, the construction of the desired constitutive expression shuttle vector can be confirmed, and the constitutive expression shuttle vector according to the present invention can be obtained even in a microorganism belonging to the genus Streptomyces. It can be said that it can be replicated even in microorganisms of the genus Escherichia.

(4) Transformant The transformant of the present invention can be prepared by transforming a host with a constitutive expression shuttle vector of the present invention or a plasmid in which a target protein gene is introduced into the constitutive expression shuttle vector. For the transformation, a conventional method may be used, for example, an electroporation method, a calcium phosphate method, or a DEAE dextran method can be used. The host used in the present invention is a microorganism of the genus Streptomyces and a microorganism of the genus Escherichia. The host used for secreting and expressing the target protein is preferably a Streptomyces microorganism. As the host used for the growth and recovery of the vector plasmid, a microorganism of the genus Streptomyces or a microorganism of the genus Escherichia can be used, but a microorganism of the genus Escherichia is preferable.

(5) Method for Producing Protein The present invention can also provide a method for producing a target protein. That is, the target protein can be produced by culturing the transformant of (4) above and collecting the target protein from the obtained culture. As the method for culturing the transformant, a method suitable for the microorganism of the genus Streptomyces used as the host may be appropriately selected.

Further, in the method for producing a protein according to the present invention, the "culture" means a product obtained by culturing a culture solution, a culture supernatant, cells, a cell-free extract, a cell membrane, or the like. In the cell-free extract, the cultured cells are physically crushed with a homogenizer or the like by adding, for example, sodium phosphate buffer, and then centrifuged (for example, 15,000 rpm, 10 min, 4 ° C.) to crush the cells that cannot be crushed. The supernatant can be collected and obtained so that (cells) are not present. The cell membrane can be obtained by suspending the pellet obtained by centrifugation in a lysis buffer.

As the target protein, the culture may be used as it is, or a known method such as dialysis or sulfur precipitation, electrophoresis, or various chromatography such as gel filtration, ion exchange, affinity, etc. may be used alone or in combination as appropriate. Therefore, concentrated and purified products may be used.

3. 3. Constitutive secretion expression shuttle vector according to the present invention The constitutive secretion expression shuttle vector according to the present invention is a DNA region capable of expressing a target protein without adding an inducer during culturing using a microorganism of the genus Streptomyces as a host. It includes a DNA region capable of secreting and expressing a target protein during culture using a Seth microorganism as a host, a DNA region capable of replicating using a Streptomyces microorganism as a host, and a DNA region capable of replicating using an Escherichia microorganism as a host. That is, the constitutive secretion expression shuttle vector according to the present invention is a shuttle vector plasmid that can replicate in both Streptomyces microorganisms and Escherichia microorganisms, and secretes and expresses the target protein in Streptomyces microorganisms. It is an effective vector to make it.

Here, in the constitutive secretion expression shuttle vector according to the present invention, a DNA region capable of expressing a target protein without adding an inducer during culturing using the Streptomyces microorganism as a host, and a Streptomyces microorganism as a host. Since the DNA region capable of secreting and expressing the target protein during culturing and the DNA region capable of replicating using a microorganism of the genus Streptomyces as a host are the same as those provided by the above-mentioned constitutive secretion expression vector according to the present invention. , These explanations are omitted here. Further, since the DNA region capable of replicating using a microorganism of the genus Escherichia as a host is the same as that provided by the above-mentioned constitutive expression shuttle vector according to the present invention, description thereof will be omitted here.

(1) Construction of Constitutive Secretory Expression Shuttle Vector As a method for constructing the constitutive secretion expression shuttle vector of the present invention, first, the signal peptide is introduced upstream of the MCS of the vector pHSA81 by the same method as described above. , To construct a constitutive secretion expression vector according to the present invention.
Then, the constructed constitutive secretion expression vector is shuttled by the same method as the method for constructing the constitutive expression shuttle vector according to the present invention.
Since the method for constructing the constitutive secretion expression vector and the method for shuttle formation are the same as those described above, the description thereof will be omitted here.

(2) plasmid Stability Test Furthermore, it is necessary to test the stability of the plasmid for the constitutive secretion expression shuttle vector according to the present invention in order to confirm the shuttle formation. The stability test is performed by the same method as the stability test for the constitutive expression shuttle vector according to the present invention. Therefore, the description of the method of this test is omitted here.

(3) Transformant The transformant of the present invention can be prepared by transforming a host with a constitutive secretion expression shuttle vector of the present invention or a plasmid in which a target protein gene is introduced into the constitutive secretion expression shuttle vector. For the transformation, a conventional method may be used, for example, an electroporation method, a calcium phosphate method, or a DEAE dextran method can be used. The host used in the present invention is a microorganism of the genus Streptomyces and a microorganism of the genus Escherichia. The host used to express the protein of interest is preferably a Streptomyces microorganism. As the host used for the growth and recovery of the vector plasmid, a microorganism of the genus Streptomyces or a microorganism of the genus Escherichia can be used, but a microorganism of the genus Escherichia is preferable.

(4) Method for Producing Protein The present invention can also provide a method for producing a target protein. That is, the target protein can be produced by culturing the transformant of (3) above and collecting the target protein from the obtained culture. As the method for culturing the transformant, a method suitable for the microorganism of the genus Streptomyces used as the host may be appropriately selected.

Further, in the method for producing a protein according to the present invention, the "culture" means a product obtained by culturing a culture solution, a culture supernatant, cells, a cell-free extract, a cell membrane, or the like. In the cell-free extract, the cultured cells are physically crushed with a homogenizer or the like by adding, for example, sodium phosphate buffer, and then centrifuged (for example, 15,000 rpm, 10 min, 4 ° C.) to crush the cells that cannot be crushed. The supernatant can be collected and obtained so that (cells) are not present. The cell membrane can be obtained by suspending the pellet obtained by centrifugation in a lysis buffer.

As the target protein, the culture may be used as it is, or a known method such as dialysis or sulfur precipitation, electrophoresis, or various chromatography such as gel filtration, ion exchange, affinity, etc. may be used alone or in combination as appropriate. Therefore, concentrated and purified products may be used.

Hereinafter, the present invention will be described in more detail based on Examples. It should be noted that the examples described below show an example of a typical example of the present invention, and the scope of the present invention is not narrowly interpreted by this.

4. Construction of a constitutive secretory expression vector (1) Construction of a reporter plasmid for signal peptide screening In order to secrete and express a protein, an N-terminal region called a signal peptide is required. Therefore, first, a signal peptide having a stronger secretory capacity was screened.
Leucine aminopeptidase (sgLAP), a secretory enzyme derived from Streptomyces griseus, was selected as the screening reporter protein. This enzyme is derived from the same genus actinomycete and is known to be a secretory enzyme secreted by the Sec pathway, and the enzyme activity can be easily measured by using a color-developing substrate. Therefore, S. lividans It is considered that heterologous secretion is easily expressed in the TK24 strain.

First, in order to use sgLAP as a reporter protein, the msgLAP gene designed to be expressed intracellularly as a mature form by removing the DNA region (signal sequence) encoding the signal peptide, and sgLAP with the original signal sequence. The genes were introduced into the constitutive expression vector pHSA81, respectively.
A PCR reaction was performed using the S. griseus genome as a template, and the sgLAP gene and msgLAP gene fragments were amplified. Part of the solution after the PCR reaction was subjected to agarose gel electrophoresis, and amplification of msgLAP and sgLAP gene fragments was confirmed.

Here, the primers used for gene amplification are as follows (SEQ ID NOs: 1 to 3).

Forward primer for sgLAP gene fragment (pHSA81 / spSGLAPct-F: SEQ ID NO: 1)
GATGAAAGGAATGAGCATatgagaccgaaccgcttctccctgc

Forward primer for msgLAP gene fragment (pHSA81 / mSGLAPct-F: SEQ ID NO: 2)
GATGAAAGGAATGAGCATATGgccggcgccgcggcc

Reverse primer for sgLAP gene fragment and msgLAP gene fragment (pHSA81 / SGLAPct-R: SEQ ID NO: 3)
CCGCTTTTGCGGGGATCTAGAtcaggtgggcggttcgccgg

Then, each amplified fragment was ligated to pH SA81 treated with restriction enzymes Nde I and Xba I (Takara Bio Inc.) by a recombination reaction using GENEART (R) Seamless Cloning and Assembly Kit (Life Technologies). .. Furthermore, pHSA81-msgLAP and pHSA81-sgLAP were constructed by transforming S. lividans TK24 by the PEG method.

(2) Confirmation of secretory expression The S. lividans TK24 strain, which retains pHSA81-sgLAP and pHSA81-msgLAP constructed by the above method and pHSA81 as a control, respectively, was cultured in YEME / tsr solid medium at 28 ° C. for 5 days. .. Furthermore, the cells were inoculated into a 10 ml YEME / tsr liquid medium in a 2 mm square, and cultured with shaking at 28 ° C. and 130 rpm for 7 days. Then, 1 ml of the culture solution was collected by centrifugation, and the culture supernatant was collected. The precipitated cells were suspended in 1 ml 10 mM KPB (pH 7.5) and then crushed by a handy sonicator. The supernatant obtained by centrifuging the crushed solution was used as a cell-free extract (CFE), and the precipitate suspended in 1 ml 10 mM KPB (pH 7.5) was used as an insoluble fraction solution.
Then, 20 μl of the culture supernatant and 8 μl of CFE were subjected to SDS-PAGE, respectively, and the presence or absence of expression was examined. The results are shown in FIG.

As shown in FIG. 2, a band of msgLAP was confirmed in the culture supernatant of pHSA81-sgLAP / S. lividans TK24. It is considered that this is because sgLAP is expressed with a signal peptide and is secreted extracellularly by being recognized by the secretory pathway.
On the other hand, in pHSA81-msgLAP / S. lividans TK24, only a small amount of msgLAP band was confirmed in the culture supernatant, and msgLAP band was confirmed in CFE. This suggests that msgLAP is expressed intracellularly in a mature state without a signal peptide and is not secreted extracellularly.
As described above, msgLAP from which the signal peptide was removed was not secreted extracellularly but accumulated intracellularly, and thus it was considered that msgLAP could be used as a reporter protein.

(3) Screening of signal peptide A signal peptide was screened using the reporter plasmid for signal peptide screening constructed by the above method. As a screening source, S. avermitilis K139 was used because it is a microorganism of the genus Streptomyces, the entire genome sequence is determined, and the total secretory protein can be predicted. Then, from the total secretory proteins from S. avermitilis K139, those predicted to depend on the Tat secretion pathway were selected, and 21 were further based on the D value calculated by the signal peptide prediction server SignalP4.0. Signal peptides were selected (see Table 1)

Here, as shown in Table 1, the DNA sequence (signal sequence) encoding each signal peptide has four digits following the predicted ORF number "SAV -..." of S. avermitilis, which is derived after ss. Take the number of and call it ss ####.

Then, a PCR reaction was carried out using the genome of the S. avermitilis K139 strain as a template, and 21 signal sequence fragments were amplified respectively. The region amplified as a signal sequence was a region containing one residue of amino acid on the C-terminal side from the Cleavage Site in addition to the sequence predicted by signalP4.0.

Here, the primers used for amplification of each signal sequence are as shown in Table 1 (SEQ ID NOs: 4 to 45).

Then, by a recombination reaction using GENEART, a DNA fragment containing each amplified signal sequence was ligated with pHSA81-msgLAP treated with the restriction enzyme Nde I (Takara Bio Inc.). As a result, each signal sequence and the msgLAP gene are linked in frame, so that a plasmid expressing the fusion protein of each signal peptide and msgLAP could be constructed.

S. lividans TK24 was transformed using each plasmid constructed in this manner, and the LAP activity in the culture supernatant of each transformant was measured and compared.
Specifically, each cell is sufficiently grown on a YEME / tsr solid medium, inoculated into a 10 ml YEME / tsr liquid medium in 2 mm squares, and shake-cultured at 28 ° C. and 130 rpm for 7 days. went. After adding 1 ml of 10 mM KPB (pH 7.5) to 1 ml of the culture solution, the bacteria were precipitated by centrifugation, and the culture supernatant was collected. Each type of transformant was cultured. Then, the culture supernatant was prepared by centrifuging the culture solution, and the LAP activity was measured. The result is shown in FIG.

From the results shown in FIG. 3, 20 μl of each sample was further subjected to SDS-PAGE, and the presence or absence of the sgLAP band was examined. Specifically, SDS-PAGE of samples of three signal sequences with high LAP activity (ss1053, ss5891 and ss1857) and three signal sequences with low LAP activity (ss1807, ss7421 and ss6215) in the culture supernatant was used. A comparison was made. The results are shown in FIG.

As shown in FIG. 4, ss1053 showed the strongest activity in each signal sequence, and its value was equivalent to the activity of the original sgLAP.
From this result, it was considered that there may be a compatibility between the target protein to be secreted and the signal peptide.

(4) Construction of constitutive secretion expression vector Based on the results shown in Fig. 4, it was considered that there might be a compatibility between the target protein to be secreted and the signal peptide, so the specific activity was relatively high 8 A constitutive secretory expression vector was constructed using these signal peptides as the signal peptides used in the secretory expression vector.

Specifically, eight signal sequences selected based on the results of the screening were introduced upstream of the multicloning site of the vector pHSA81 to construct a constitutive secretion expression vector.
First, a PCR reaction was performed using the genome of the S. avermitilis K139 strain as a template, and eight signal sequence fragments were amplified respectively.

Here, the primers used for amplifying the eight signal sequences are as shown in Table 2 (SEQ ID NOs: 46 to 61).

Then, each amplified DNA fragment was ligated to the vector pHSA81 treated with the restriction enzyme Nde I (Takara Bio Inc.) by a recombination reaction using GENEART.
S. lividans TK24 was transformed with each GENEART reaction solution by the PEG method to obtain colonies. The resulting colonies were picked and subcultured in YEME / tsr solid medium. The cells sufficiently grown on the subcultured YEME / tsr solid medium were inoculated into 10 ml YEME / tsr liquid medium, and shake-cultured at 28 ° C. and 130 rpm for 4 days. Bacterial cells were collected from the culture solution by centrifugation, and a plasmid was extracted. DNA sequence analysis was performed on these to confirm that the target fragment was introduced upstream of the Nde I recognition site and that no mutation was contained, and that eight constitutive secretion expression vectors (pH SA81-ss1053) were confirmed. , PHSA81-ss1857, pHSA81-ss3789, pHSA81-ss5891, pHSA81-ss6579, pHSA81-ss7089, pHSA81-ss7207, pHSA81-ss7442).

For example, in the constitutive secretion expression vector pHSA81-ss1053, a signal sequence starting from ATG is placed under the strong promoter of H-NHase, and MCS starting from the Nde I recognition site is placed behind it. Since the signal sequence portion does not contain a sequence, the Nde I site in MCS is the only site in this secretory expression vector. Using this Nde I site, the target gene can be introduced from the start codon ATG.
Then, when transcribed and translated, a fusion protein of a signal peptide and a target protein is expressed. This signal peptide is recognized in the secretory pathway, and the target protein is secreted extracellularly. The signal peptide is processed during secretion, and the N-terminal of the secreted target protein is a total of histidine and methionine derived from the Nde I recognition site ATGCAT, in addition to alanine located on the C-terminal side of one residue from the Cleavage Site of the signal peptide. It is expected that 3 residues will remain at the N-terminus.

(5) Functional analysis of constitutive secretion expression vector Of the eight constitutive secretion expression vectors constructed, the functional analysis was performed using the vector pHSA81-ss1053.
Specifically, the expression of pulA (Pullulanase), PP3812 (Esterase), chiH (Chitinase), pla2 (Phospholipase A2), and apkA (Amylase) were confirmed. The method for confirming the expression of each enzyme is as follows.

(A) Confirmation of expression of pullA (Pullulanase)
Using the genomic DNA of Klebsiella aerogenes W70 as a source, PCR was performed using primers 81ss1053 (/ Nde) -mpulA_F (SEQ ID NO: 62 below) and pHSA81 (/ Xba) -pulA_R (SEQ ID NO: 63 below).
Furthermore, the amplified DNA fragment was ligated to the vector pHSA81-ss1053 cleaved with restriction enzymes NdeI and XbaI (Takara Bio Inc.) to construct pHSA81-ss1053-pulA using actinomycete S. lividans TK24 as a host. As a control, S. lividans TK24 was transformed with the vector pHSA81-ss1053.

81ss1053 (/ Nde)-mpulA_F (SEQ ID NO: 62)
TCGGCCGAGGCCGCGCATATGtgtgataacagctcttcctcttctacctctgg

pHSA81 (/ Xba)-pulA_R (SEQ ID NO: 63)
CCGCTTTTGCGGGGATCTAGAttatttactgctcaccggcaggccag

After pre-culturing each transformant at 28 ° C for 4 days using 10 ml YEME / Tsr medium, the culture solution having a wet cell weight of 2 mg was subcultured for 7 days, and the culture supernatant was prepared by centrifugation. Then, the enzyme activity was measured.
As a method for measuring enzyme activity, a reaction was carried out at 40 ° C for 5 min using Pullulan as a substrate, and the amount of reduced sugar decomposed by Pullulanase was quantified using the Somogyi-Nelson method. As a result, pHSA81-ss1053-pulA The specific activity of / S. lividans TK24 was 1.36 units / mg, and the specific activity of pHSA81-ss1053 / S. lividans TK24 was 0.0351 units / mg.

Furthermore, the prepared culture supernatant was subjected to SDS-PAGE. The result is shown in FIG. As shown in FIG. 5, a band of 117 kDa was confirmed only in pHSA81-ss1053-pulA / S. Lividans TK24. In addition, a plate assay was performed using each transformant. The result is shown in FIG. The medium composition used for the plate assay is 2YT + Red pullulan (substrate). As shown in FIG. 6, as a result of culturing at 28 ° C. for 7 days, halo formation was observed only in pHSA81-ss1053-pulA / S. Lividans TK24. From the above, it was confirmed that Pullulanase was secreted extracellularly.

(B) Confirmation of expression of PP3812 (Esterase)
Using the genomic DNA of Pseudomonas putida KT2440 as a source, PCR was performed using primers 81ss1053 (/ Nde) -mPP3812_F (SEQ ID NO: 64 below) and pHSA81 (/ Xba) -PP3812_R (SEQ ID NO: 65 below).
Furthermore, the amplified DNA fragment was ligated to pHSA81-ss1053 cleaved with restriction enzymes NdeI and XbaI (Takara Bio Inc.) to construct pHSA81-ss1053-PP3812 using actinomycete S. lividans TK24 as a host. S. lividans TK24 was transformed with pHSA81-ss1053 as a control.
After pre-culturing each transformant at 28 ° C for 4 days using 10 ml YEME / Tsr medium, the culture solution having a wet cell weight of 2 mg was subcultured for 7 days, and the culture supernatant was prepared by centrifugation. Then, the enzyme activity was measured. The enzyme activity is measured by reacting at 30 ° C using p-Nitrophenyl acetate as a substrate, measuring the absorption at 405 nm every 2 sec up to 60 sec, and using the produced p-Nitrophenol as the molar absorption of p-Nitrophenol. Quantified from the coefficient.
As a result, the specific activity of pHSA81-ss1053-PP3812 / S. Lividans TK24 was 0.489 units / mg, and the specific activity of pHSA81-ss1053 / S. Lividans TK24 was 0 units / mg.

81ss1053 (/ Nde)-mPP3812_F (SEQ ID NO: 64)
TCGGCCGAGGCCGCGCATATGgcaggcagccctggtgtcgaac

pHSA81 (/ Xba)-PP3812_R (SEQ ID NO: 65)
CCGCTTTTGCGGGGATCTAGAtcactgcagatgcactttaagttcgttgcc

Furthermore, when the prepared culture supernatant was subjected to SDS-PAGE, a band of 34.1 kDa was confirmed only in pHSA81-ss1053-PP3812 / S. lividans TK24 (see FIG. 5). From the above, the expression of extracellular secretion of esterase was confirmed.

(C) Confirmation of expression of chiH (Chitinase)
PCR was performed using the primers 81ss1053 (/ Nde) -mchiH_F (SEQ ID NO: 66 below) and pHSA81 (/ Xba) -chiH_R (SEQ ID NO: 67 below) from the genomic DNA of Streptomyces coelicolor A3 (2).
Furthermore, the amplified DNA fragment was ligated to pHSA81-ss1053 cleaved with restriction enzymes NdeI and XbaI (Takara Bio Inc.) to construct pHSA81-ss1053-chiH using actinomycete S. lividans TK24 as a host. S. lividans TK24 was transformed with pHSA81-ss1053 as a control.
Each transformant was pre-cultured at 28 ° C for 4 days using 10 ml YEME / Tsr medium, and then the culture solution having a wet cell weight of 2 mg was subcultured for 7 days, and the culture supernatant was prepared by centrifugation. Then, the enzyme activity was measured.
The enzyme activity was measured by reacting 4-Methylumbelliferyl- (GlcNAc) ₃ as a substrate at 37 ° C for 10 min and measuring the fluorescence of the produced 4-Methylumbelliferone (excitation: 360 nm, measurement: 450 nm). Quantified.
As a result, the specific activity of pHSA81-ss1053-chiH / S. Lividans TK24 was 4.24 units / mg, and the specific activity of pHSA81-ss1053 / S. Lividans TK24 was 0.181 units / mg.

81ss1053 (/ Nde)-mchiH_F (SEQ ID NO: 66)
TCGGCCGAGGCCGCGCATATGgcgaccccgctgccgga

pHSA81 (/ Xba)-chiH_R (SEQ ID NO: 67)
CCGCTTTTGCGGGGATCTAGAtcagcaggcgccgaggtcct

Furthermore, when the prepared culture supernatant was subjected to SDS-PAGE, a band of 49.5 kDa was confirmed only in pHSA81-ss1053-chiH / S. Lividans TK24 (see FIG. 5).
In addition, a plate assay was performed using each transformant. The medium composition used in the plate assay is Colloidal chitin single carbon source medium. As shown in FIG. 6, as a result of culturing at 28 ° C. for 7 days, halo formation was observed only in pHSA81-ss1053-chiH / S. Lividans TK24. From the above, it was confirmed that chitinase was secreted extracellularly.

(D) Confirmation of expression of pla2 (Phospholipase A2)
Using the genomic DNA of S. coelicolor A3 (2) as a source, PCR was performed using primers 81ss1053 (/ Nde) -mplaA2_F (SEQ ID NO: 68 below) and pHSA81 (/ Xba) -plaA2_R (SEQ ID NO: 69 below).
Furthermore, the amplified DNA fragment was ligated to pHSA81-ss1053 cleaved with restriction enzymes NdeI and XbaI (Takara Bio Inc.) to construct pHSA81-ss1053-pla2 using actinomycete S. lividans TK24 as a host. S. lividans TK24 was transformed with pHSA81-ss1053 as a control.
Each transformant was pre-cultured at 28 ° C for 4 days using 10 ml YEME / Tsr medium, and then the culture solution having a wet cell weight of 2 mg was subcultured for 7 days, and the culture supernatant was prepared by centrifugation. Then, the enzyme activity was measured. The enzyme activity was measured using the sPLA ₂ Assay Kit (Cayman Chemical Company).
As a result, the specific activity of pHSA81-ss1053-pla2 / S. Lividans TK24 was 8.13 units / mg, and that of pHSA81-ss1053 / S. Lividans TK24 was not observed.

81ss1053 (/ Nde)-mplaA2_F (SEQ ID NO: 68)
TCGGCCGAGGCCGCGCATATGgctcccgccgacaaggcaca

pHSA81 (/ Xba)-plaA2_R (SEQ ID NO: 69)
CCGCTTTTGCGGGGATCTAGAtcagccgaacaccttcacggcct

Furthermore, when the prepared culture supernatant was subjected to SDS-PAGE, a 14 kDa band was confirmed only in pHSA81-ss1053-pla2 / S. lividans TK24 (see FIG. 5). From the above, it was confirmed that Phospholipase A2 was secreted extracellularly.

(E) Confirmation of expression of apkA (Amylase)
PCR was performed using the primers 81ss1053 (/ Nde) -mapkAH_F (SEQ ID NO: 70 below) and pHSA81 (/ Xba) -apkA_R (SEQ ID NO: 71 below) from the genomic DNA of Thermococcus kodakaraensis KOD1.
Furthermore, the amplified DNA fragment was ligated to pHSA81-ss1053 cleaved with restriction enzymes NdeI and XbaI (Takara Bio Inc.) to construct pHSA81-ss1053-apkA using actinomycete S. lividans TK24 as a host. S. lividans TK24 was transformed with pHSA81-ss1053 as a control.
Each transformant was pre-cultured at 28 ° C for 4 days using 10 ml YEME / Tsr medium, and then the culture solution having a wet cell weight of 2 mg was subcultured for 7 days, and the culture supernatant was prepared by centrifugation. Then, the enzyme activity was measured. As a method for measuring enzyme activity, Starch was used as a substrate and a reaction was carried out at 40 ° C. for 5 min, and the amount of reducing sugar decomposed by Amylase was quantified using the Somogyi-Nelson method.
As a result, the specific activity of pHSA81-ss1053-apkA / S. Lividans TK24 was 5.32 units / mg, and the specific activity of pHSA81-ss1053 / S. Lividans TK24 was 0.172 units / mg.

81ss1053 (/ Nde)-mapkA_F (SEQ ID NO: 70)
TCGGCCGAGGCCGCGCATATGgcaaagtattccgaactcgaagaaggcgg

pHSA81 (/ Xba)-apkA_R (SEQ ID NO: 71)
CCGCTTTTGCGGGGATCTAGAtcatccaaccccgcagtagctccag

In addition, a plate assay was performed using each transformant. The medium composition used for the plate assay is 2YT + Starch medium. As shown in FIG. 6, a plate cultured at 28 ° C. for 7 days was incubated at 60 ° C. for about 3 hours, and then a diluted iodine solution was directly sprinkled on the plate to carry out an iodine-starch reaction. As a result, halo formation was observed only in pHSA81-ss1053-apkA / S. Lividans TK24. From the above, it was confirmed that Amylase was secreted extracellularly.

5. Construction of constitutive expression shuttle vector (1) Construction of constitutive expression shuttle vector First, the starting point of plasmid replication of Escherichia coli and antibiotic resistance genes for Escherichia coli microorganisms (kanamycin resistance gene Km, chloramphenicol resistance gene Cm, ampicillin resistance) A DNA fragment containing the gene Ap) was amplified by PCR.
In this case, pHSA81 (BlpI) -ori_F (SEQ ID NO: 72 below) and pHSA81 (BlpI) -ori_KmR (F) _R (SEQ ID NO: 73 below) were used as primers, and the kanamycin resistance gene Km was used as the H-NHase gene promoter. The DNA fragment ori_Km (F) introduced in the same direction was amplified.
As primers, pHSA81 (BlpI) -ori_KmR (R) _F (SEQ ID NO: 74 below) and pHSA81 (BlpI) -ori_R (SEQ ID NO: 75 below) were used, and the kanamycin resistance gene Km was reversed from that of the H-NHase gene promoter. The DNA fragment ori_Km (R) introduced in the direction was amplified.
As primers, pHSA81 (BlpI) -ori_F (SEQ ID NO: 72 below) and pHSA81 (BlpI) -ori_CmR (F) _R (SEQ ID NO: 76 below) were used, and the chloramphenicol resistance gene Cm was the H-NHase gene. The DNA fragment ori_Cm (F) introduced in the same direction as the promoter was amplified.
As primers, pHSA81 (BlpI) -ori_CmR (R) _F (SEQ ID NO: 77 below) and pHSA81 (BlpI) -ori_R (SEQ ID NO: 75 below) were used, and the chloramphenicol resistance gene Cm was the H-NHase gene. The DNA fragment ori_Cm (R) introduced in the opposite direction to the promoter was amplified.
As primers, pHSA81 (BlpI) -ori_ampR (F) _F (SEQ ID NO: 78 below) and pHSA81 (BlpI) -ori_R (SEQ ID NO: 75 below) are used, and the ampicillin resistance gene Ap is the same as the H-NHase gene promoter. The DNA fragment ori_Ap (F) introduced in the direction was amplified.
As primers, pHSA81 (BlpI) -ori_F (SEQ ID NO: 72 below) and pHSA81 (BlpI) -ori_ampR (R) _R (SEQ ID NO: 79 below) were used, and the ampicillin resistance gene Ap was reversed from that of the H-NHase gene promoter. The DNA fragment ori_Km (R) introduced in the direction was amplified.
Here, the primers used for amplifying the DNA fragment and the extended DNA fragment are as follows (SEQ ID NOS: 72 to 79). The templates used for amplification of each DNA fragment are shown in Table 3 below.

pHSA81 (BlpI) -ori_F (SEQ ID NO: 72)
CCCAGTCGCAACCGGGCTCAGCacatgtgagcaaaaggccagcaaaaggc

pHSA81 (BlpI) -ori_KmR (F) _R (SEQ ID NO: 73)
GGTCCGCCCACTGCGCTGAGCtcgatttattcaacaaagccgccgtccc

pHSA81 (BlpI) -ori_KmR (R) _F (SEQ ID NO: 74)
CCCAGTCGCAACCGGGCTGAGCtcgatttattcaacaaagccgccgtccc

pHSA81 (BlpI) -ori_R (SEQ ID NO: 75)
GGTCCGCCCACTGCGCTCAGCacatgtgagcaaaaggccagcaaaaggc

pHSA81 (BlpI) -ori_CmR (F) _R (SEQ ID NO: 76)
GGTCCGCCCACTGCGCTGAGCtcgaatttctgccattcatccgcttattatcacttattcag

pHSA81 (BlpI) -ori_CmR (R) _F (SEQ ID NO: 77)
CCAGTCGCAACCGGGCTGAGCtcgaatttctgccattcatccgcttattatcacttattcag

pHSA81 (BlpI)-ori_ampR (F) _F (SEQ ID NO: 78)
CCCAGTCGCAACCGGGCTGAGCgacgtcaggtggcacttttcgggg

pHSA81 (BlpI) -ori_ampR (R) _R (SEQ ID NO: 79)
GGTCCGCCCACTGCGCTGAGCgacgtcaggtggcacttttcgggg

Next, the amplified DNA fragment was ligated to the vector pHSA81 treated with the restriction enzyme BlpI (Takara Bio Inc.) by a recombination reaction using GENEART. Furthermore, E. coli TOP10 and Escherichia coli stbl2 were transformed by the heat shock method using each GENEART reaction solution to obtain colonies. The obtained colonies were picked and subcultured in 2YT solid medium containing each drug.
As a preculture, each cell grown on a 2YT solid medium was inoculated into a 5 ml 2YT liquid medium, and cultured at 37 ° C. and 150 rpm until the medium became sufficiently turbid (about 12 hours). .. The plasmid was extracted from the cells after culturing.
From the preculture, 1% inoculation was carried out in a new 5 ml 2YT liquid medium, and the culture was carried out again for 12 hours. This was subcultured again, and after the third subculture, the cells were collected and the plasmid was extracted.

S. lividans TK24 was transformed with each plasmid by the PEG method to obtain colonies.
Each of the cells sufficiently grown on the YEME / tsr solid medium was inoculated into a 10 ml YEME / tsr liquid medium in 2 mm squares, and cultured with shaking at 28 ° C. and 130 rpm for 4 days. Subculture the culture solution into a new 10 ml YEME / tsr liquid medium culture solution with 1% inoculation, extract the plasmid from the cells subjected to a total of 3 subcultures, and constructive expression shuttle vectors (pEHSA81k, pEHSA81k). (r), pEHSA81c, pEHSA81c (r), pEHSA81a, pEHSA81a (r)) were obtained. The composition of each constitutive expression shuttle vector is shown in FIG.
In FIG. 7, the vector in which the DNA fragment ori_Km (F) is linked to pHSA81 is pEHSA81k, the vector in which the DNA fragment ori_Km (R) is linked is pEHSA81k (r), and the vector in which the DNA fragment ori_Cm (F) is linked. Is pEHSA81c, the vector ligating the DNA fragment ori_Cm (R) is pEHSA81c (r), the vector ligating the DNA fragment ori_Ap (F) is pEHSA81a, and the vector ligating the DNA fragment ori_Ap (R) is pEHSA81a (r). ing.

(2) Stability test of each constitutive expression shuttle vector Each plasmid after 3 subcultures with Escherichia coli or 3 subcultures with actinomycetes is treated with the restriction enzyme BlpI and subjected to agarose gel electrophoresis. did. Then, the stability of the plasmid was examined from the band pattern of the DNA fragment.
As a result, it was confirmed that pHSA81a and pHSA81a (r) were stably retained without being deficient when Escherichia coli stbl2 and TOP10 were used as hosts. The host is not limited to these strains as long as the host is stably retained without loss of the vector.
It was confirmed that pHSA81c and pHSA81c (r) were deficient when Escherichia coli TOP10 was used as a host, but were stably retained without being deficient when Escherichia coli stbl2 was used as a host. The host is not limited to these strains as long as the host is stably retained without loss of the vector.
It was confirmed that pHSA81k and pHSA81k (r) were deficient when Escherichia coli stbl2 was used as a host, but were stably retained without being deficient when Escherichia coli TOP10 was used as a host. The host is not limited to these strains as long as the host is stably retained without loss of the vector.
In addition, each plasmid (pEHSA81k, pEHSA81k (r), pEHSA81c, pEHSA81c (r), pEHSA81a, pEHSA81a (r)) after the first liquid culture in E. coli was deleted regardless of whether E. coli stbl2 or TOP10 was used as the host. It was confirmed that it was held stably without any trouble.
On the other hand, in the plasmid after three subcultures in S. lividans TK24, no band other than the target was observed, and it was confirmed that the plasmid was stably retained in S. lividans TK24.
Since it was confirmed that all plasmids were stably retained in S. lividans TK24, it can be inferred that these plasmids have a function as a constitutive expression shuttle vector.

(3) Functional analysis of the constitutive expression shuttle vector Next, Green Fluorescent Protein (GFP) was integrated as a reporter gene into the constitutive expression shuttle vectors pEHSA81k, pEHSA81c and pEHSA81a, and the expression in S. lividans TK24 was subjected to SDS-PAGE. The functionality was examined by confirming with. As a control, each reporter gene was incorporated into the vector pHSA81 and compared.
In addition, leucine aminopeptidase (sgLAP), which is a secretory enzyme derived from S. griseus, and catechol 2,3-dioxygenase gene (xylE) derived from Pseudomonas putida were integrated into the constitutive expression shuttle vector pEHSA81k as a reporter gene, and S. The functionality of lividans TK24 was also examined by confirming its expression on SDS-PAGE. As a control, each reporter gene was incorporated into the vector pHSA81 and compared.

(A) Construction and expression results of a vector containing GFP First, GFP was amplified by PCR using pAcGFP (Takara Bio Inc.) as a template. Next, the DNA fragments obtained by PCR were treated with restriction enzymes XbaI and SacI (Takara Bio Inc.), and the In-Fusion HD Cloning Kit (Takara Bio Inc.) was used for each constitutive expression shuttle vector and vector pHSA81. PEHSA81k-GFP, pEHSA81c-GFP, and pEHSA81a-GFP were constructed using E. coli TOP10 as a host, and pHSA81-GFP was constructed using S. lividans TK24 as a host.

Then, S. lividans TK24 was transformed with the constructed plasmid. After culturing, a cell-free extract was prepared, and the fluorescence of GFP was measured with a fluorescence measuring device. As a result, the total fluorescence intensity in 10 mL of the culture solution was 34700 for pHSA81-GFP / S. Lividans TK24, 9160 for pEHSA81k-GFP / S. Lividans TK24, and 13500 for pEHSA81c-GFP / S. Lividans TK24. The pEHSA81a-GFP / S. Lividans TK24 was 9600. Expression of GFP was confirmed in all vectors.

(B) Construction and expression results of a vector containing sgLAP First, sgLAP was amplified by PCR using S. griseus as a template. Next, the DNA fragment obtained by PCR was treated with restriction enzymes XbaI and SacI (Takara Bio Inc.) using the constitutive expression shuttle vector pEHSA81k and the vector pHSA81 using the In-Fusion HD Cloning Kit (Takara Bio Inc.). PEHSA81k-sgLAP was constructed using E. coli TOP10 as a host, and pHSA81-sgLAP was constructed using S. lividans TK24 as a host.

Then, S. lividans TK24 was transformed with the constructed plasmid. After culturing, leucine aminopeptidase in the culture supernatant was measured. As a result, pHSA81-gLAP / S. lividans TK24 was 647 units / mg and pEHSA81k-gLAP / S. lividans TK24 was 502 units / mg, and the expression levels of both were almost the same.

(C) Construction and expression results of vector containing xylE First, xylE was amplified by PCR using Pseudomonas putida (Nakai et al. 1983, Worsey and Williams. 1975) as a template.
Next, the DNA fragment obtained by PCR was ligated to the vector pEHSA81k and the vector pHSA81 treated with restriction enzymes XbaI and SacI (Takara Bio Inc.) using the In-Fusion HD Cloning Kit (Takara Bio Inc.). pEHSA81k-xylE was constructed using E. coli TOP10 as a host, and pHSA81-xylE was constructed using S. lividans TK24 as a host.
Then, S. lividans TK24 was transformed with the constructed plasmid. After culturing, a cell-free extract was prepared and the catechol 2,3-dioxygenase activity was measured. As a result, pHSA81-xylE / S. lividans TK24 was 0.56 units / mg and pEHSA81k-xylE / S. Lividans TK24 was 1.93 units / mg. The expression level of the constitutive expression shuttle vector pEHSA81k constructed this time was higher than that of pHSA81. There were many.

6. Construction of Constitutive Secretory Expression Shuttle Vector (1) Method of Constructing Constitutive Secretory Expression Shuttle Vector First, eight constitutive secretions are constructed by the same method as described in "4. Construction of Constitutive Secretion Expression Vector" above. Expression vectors (pHSA81-ss1053, pHSA81-ss1857, pHSA81-ss3789, pHSA81-ss5891, pHSA81-ss6579, pHSA81-ss7089, pHSA81-ss7207, pHSA81-ss7442) were constructed.
Next, a DNA fragment containing the plasmid replication initiation origin of Escherichia coli and the kanamycin resistance gene Km was used as a template by using the expression vector pHSA81 as a template by the same method as described in "5. Construction of a constitutive expression shuttle vector" above. Amplified by PCR.
Then, by a recombination reaction using the In-Fusion HD Cloning Kit (Takara Bio Inc.), a DNA fragment ori-rep-KmR (F) having a replication region amplified by PCR and a kanamycin resistance gene Km, and a restriction enzyme BlpI Constitutive secretion expression vectors treated with (Takara Bio Inc.) were ligated to form a constitutive secretion expression shuttle vector (pEHSA81-ss1053, pEHSA81-ss1857, pEHSA81-ss3789, pEHSA81-ss5891, pEHSA81-ss6579, pEHSA81-ss7089). , PEHSA81-ss7207, pEHSA81-ss7442). FIG. 8 shows the configuration of the constitutive secretion expression shuttle vector pEHSA81-ss1053.

Here, in the above-mentioned constitutive expression shuttle vectors (pEHSA81k, pEHSA81k (r), pEHSA81c, pEHSA81c (r), pEHSA81a, pEHSA81a (r)), the stability of the plasmid was confirmed. Also for secretory expression shuttle vectors (pEHSA81-ss1053, pEHSA81-ss1857, pEHSA81-ss3789, pEHSA81-ss5891, pEHSA81-ss6579, pEHSA81-ss7089, pEHSA81-ss7207, pEHSA81-ss7442, pEHSA81-ss7442) can.

(2) Functional analysis of the constitutive secretion expression shuttle vector Of the eight constitutive secretion expression shuttle vectors constructed, the functional analysis was performed using the vector pEHSA81-ss1053.
Specifically, the expression of pulA (Pullulanase), PP3812 (Esterase), chiH (Chitinase), pla2 (Phospholipase A2), and apkA (Amylase) were confirmed. The method for confirming the expression of each enzyme is the above-mentioned constitutive secretion expression vector according to the present invention, except that a plasmid was constructed using Escherichia coli TOP10 as a host and S. lividans TK24 was transformed using the constructed plasmid. Since it is the same as the functional analysis method of, the description thereof is omitted here.

(A) Expression result of pulA (Pullulanase)
As a result of measuring the enzymatic activity of pulA, the specific activity of pEHSA81k-ss1053-pulA / S. Lividans TK24 was 1.23 units / mg, and the specific activity of pEHSA81k-ss1053 / S. Lividans TK24 was 0.0754 units / mg. In addition, pEHSA81k-ss1053-pulA indicates pEHSA81k-ss1053- in which pulA was introduced.
Furthermore, the prepared culture supernatant was subjected to SDS-PAGE. The result is shown in FIG. As shown in FIG. 9, a band of 117 kDa was confirmed only in pEHSA81k-ss1053-pulA / S. Lividans TK24.
In addition, a plate assay was performed using the transformant. The result is shown in FIG. The medium composition used in the plate assay is 2YT + Red pullulan (substrate).
As shown in FIG. 10, as a result of culturing at 28 ° C. for 7 days, halo formation was observed only in pEHSA81k-ss1053-pulA / S. Lividans TK24. From the above, it was confirmed that Pullulanase was secreted extracellularly.

(B) Expression result of PP3812 (Esterase)
As a result of measuring the enzymatic activity of PP3812, the specific activity of pEHSA81k-ss1053-PP3812 / S. lividans TK24 was 0.816 units / mg, and the specific activity of pEHSA81k-ss1053 / S. Lividans TK24 was 0 units / mg. In addition, pEHSA81k-ss1053-PP3812 indicates pEHSA81k-ss1053- in which PP3812 was introduced.
Furthermore, when the prepared culture supernatant was subjected to SDS-PAGE, a band of 34.1 kDa was confirmed only in pEHSA81k-ss1053-PP3812 / S. lividans TK24 (see FIG. 9). From the above, the expression of extracellular secretion of esterase was confirmed.

(C) Expression result of chiH (Chitinase)
As a result of measuring the enzyme activity of chiH, the specific activity of pEHSA81k-ss1053-chiH / S. Lividans TK24 was 7.89 units / mg, and the specific activity of pEHSA81k-ss1053 / S. Lividans TK24 was 0.321 units / mg. In addition, pEHSA81k-ss1053-chiH indicates pEHSA81k-ss1053 in which chiH was introduced.
Furthermore, when the prepared culture supernatant was subjected to SDS-PAGE, a band of 49.5 kDa was confirmed only in pEHSA81k-ss1053-chiH / S. Lividans TK24 (see FIG. 9).
In addition, as a result of plate assay, halo formation was observed only in pEHSA81k-ss1053-chiH / S. Lividans TK24 (see FIG. 10). From the above, it was confirmed that chitinase was secreted extracellularly.

(D) Expression result of pla2 (Phospholipase A2)
As a result of measuring the enzyme activity of pla2, the specific activity of pEHSA81k-ss1053-pla2 / S. Lividans TK24 was 6.06 units / mg, but the enzyme activity was not observed in pEHSA81k-ss1053 / S. Lividans TK24. In addition, pEHSA81k-ss1053-pla2 indicates pEHSA81k-ss1053 in which pla2 was introduced.
Furthermore, when the prepared culture supernatant was subjected to SDS-PAGE, a 14 kDa band was confirmed only in pEHSA81k-ss1053-pla2 / S. lividans TK24 (see FIG. 9).
From the above, it was confirmed that Phospholipase A2 was secreted extracellularly.

(E) Expression result of apkA (Amylase)
As a result of measuring the enzyme activity of apkA, the specific activity of pEHSA81k-ss1053-apkA / S. Lividans TK24 was 1.54 units / mg, and the specific activity of pEHSA81k-ss1053 / S. Lividans TK24 was 0.141 units / mg. In addition, pEHSA81k-ss1053-apkA indicates pEHSA81k-ss1053 in which apkA is introduced.
Furthermore, as a result of plate assay, halo formation was observed only in pEHSA81k-ss1053-apkA / S. Lividans TK24 (see FIG. 10). From the above, it was confirmed that Amylase was secreted extracellularly.

Claims (4)

It is a constitutive secretion expression plasmid vector containing the following regions (a) to (c), and the vector is subjected to the following (b) in pHSA81 in order to secrete and express the target protein to the outside of the microorganism using the Tat secretion pathway. Constitutive secretory expression plasmid vector into which the region of
(A) A DNA region capable of expressing a target protein without adding an inducer during culturing using a microorganism of the genus Streptomyces as a host, and the DNA region is a DNA sequence encoding a regulatory protein (NhC) and a transcription activation protein. Contains a DNA sequence encoding (NhD);
(B) A DNA region capable of secreting and expressing a target protein during culturing using a microorganism of the genus Streptomyces as a host.
In the DNA region, the DNA sequence encoding the signal peptide is linked to the cut portion of the restriction enzyme NdeI site of the restriction enzyme NdeI site upstream of the multicloning site.
The signal peptide is any one of SAV1053, SAV1857, SAV3789, SAV5891, SAV6579, SAV7089, SAV7207, or SAV7442 from S. avermitilis;
(C) DNA region capable of replicating using a microorganism of the genus Streptomyces as a host A constitutive secretory expression shuttle vector for secreting and expressing the target protein outside the microorganism using the Tat secretory pathway, which comprises the following regions (g) to (j), and the vector has the following (h) at pH SA81. ) And (j) are introduced into the constitutive secretion expression shuttle vector.
(G) A DNA region capable of expressing a target protein without adding an inducer during culturing using a microorganism of the genus Streptomyces as a host, and the DNA region is a DNA sequence encoding a regulatory protein (NhC) and a transcription activation protein. Contains a DNA sequence encoding (NhD);
(H) A DNA region capable of secreting and expressing a target protein during culturing using a microorganism of the genus Streptomyces as a host.
In the DNA region, the DNA sequence encoding the signal peptide is linked to the cut portion of the restriction enzyme NdeI site of the restriction enzyme NdeI site upstream of the multicloning site.
The signal peptide is any one of SAV1053, SAV1857, SAV3789, SAV5891, SAV6579, SAV7089, SAV7207, or SAV7442 from S. avermitilis;
(I) A DNA region capable of replicating using a microorganism of the genus Streptomyces as a host;
(J) DNA region capable of replicating using a microorganism of the genus Escherichia as a host A transformant obtained by transforming a Streptomyces microorganism with the vector according to claim 1 or 2. A method for producing a target protein, which comprises culturing the transformant according to claim 3 and collecting the target protein from the obtained culture.

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