KR101349070B1 - Compulsory expression vector of foreign gene - Google Patents

Abstract

The present invention relates to a foreign gene forced expression vector, specifically, T7 promoter, lactose operator and three-frame Simultaneous Translation (TFST) construct [three ribosomal binding sites (RBS) and Three start codons (ATG) -containing fragments which are started in different frames] were sequentially constructed, and a gene-forced expression cassette including one or two gene-expression cassettes was introduced into the vector. A DNA fragment to be cloned is transcribed into one transcription unit (mRNA), from which a forced expression vector can be produced from which three or six frames of polypeptide can be translated simultaneously, and the green fluorescence in three or six frames on the vector. After cloning the protein gene, each recombinant expression vector obtained was high in all the green fluorescent protein in the transformed strain. Since it was confirmed that the expression level, the novel expression vector of the present invention can be used for the production of metagenome, genome, or cDNA library, as well as a method for expressing the active protein by preparing a metagenomic library, genomic library or cDNA library The present invention can be useful for screening useful genes from these libraries, or for discovering genes using an in vitro transcription / translation system.

Description

Compulsory expression vector of foreign gene}

The present invention relates to novel expression vectors for the search for useful genes.

The industrial use of enzymes produced from microorganisms has been extensively conducted in various fields for the last 100 years. These studies were mainly conducted on microorganisms that can be cultured, and various microbial enzymes found therefrom are being used industrially.

However, recent studies of molecular microbial ecology have demonstrated that more than 99% of the microorganisms in nature are not cultured in typical media (Amann et al. al . , Microbiol. Rev. 59: 143-169, 1995; Hugenholtz and Pace, Trends Biotechnol. 14: 190-197, 1996; Ward et al . , Nature 345: 63-65, 1990). Therefore, efforts are needed to find enzymes from microorganisms that are not yet cultivable and identified, which is believed to be possible through molecular biological recombinant expression.

As part of this approach, research on metagenome has been attempted, which refers to the genomes of all microorganisms in nature, and in general, metagenome studies do not incubate microorganisms from natural samples. After separating the genomes, they are prepared into a library and introduced into cultivable Escherichia coli. Although it is difficult to interpret the entire genome information for the cultivated microorganisms, there is an advantage that can secure the gene source product derived from the microorganism.

The process of extracting metagenomes from environmental samples to build libraries and screening for useful genes began in the mid-1990s. The most typical screening method involves extracting metagenome from environmental samples such as soil and seawater, inserting them into bacterial artificial chromosome (BAC), fosmid, or plasmid vectors to transform E. coli , It is a method of viewing activity on a flat plate containing. To date, gene genomes related to antibiotic synthesis and antibiotic resistance, as well as agarase, amidase, amylase, esterase, lipase and xyl It has been reported that enzyme genes used in industry such as lanase, pectate lyase and polyketide synthases have been screened (Daniel, D. Curr. Opinion Biotechnol. 15: 199-204) , 2004; Streit, WR et al . , Curr. Opinion Biotechnol. 15: 285-290, 2004; Yun, J. and S. Ryu, Microbial. Cell Factories 4: 8, 2005).

Recently, methods for improving the above methods have been studied in building and screening the metagenome library. First, the host E. coli was used to recognize the limitations of transcription and translation system involvement in expressing metagenome genes derived from various microorganisms in environmental samples as active proteins, and in addition to E. coli , streptomy Streptomyces lividans or Pseudomonas putida ) has also been reported to build a metagenome library using the host (Courtois, S. et. al . , Appl. Environ. Microbiol. 69: 49-55, 2003; Martinez, A. et al . , Appl. Environ. Microbiol. 70: 2452-2463, 2004; Wang, GYS et al . , Org. Lett. 2: 2401-2404, 2000). In addition, a method of pre-enrichment of the library in a medium containing the substrate has been attempted to screen specific enzymes (Entcheva, P. et. al . , Appl. Environ Microbiol. 67: 89-99, 2001; Gabor, EM et al . , Environ. Microbiol. 6: 948-958, 2004a; Knietsch, A. et al . , Appl. Environ. Microbiol. 69: 1408-1416, 2003; Voget, S. et al . , Appl. Environ. Microbiol. 69: 6235-6242, 2003). In addition, a method (SIGEX) for screening genes induced by expression of a desired substrate in a metagenome library in liquid culture (SIGEX) has been reported (Uchiyama, T. et. al . , Nat. Biotechnol. 23: 88-93, 2005). In addition, a bioinformatics approach has been used to verify the stochastic utility of finding desired genes in the metagenome library (Gabor, EM et al. al . , Environ. Microbiol. 6: 879-886, 2004b). In addition, after constructing the metagenome library, shotgun sequencing is performed to randomly reveal DNA information of bacteria that cannot be cultured (Tyron, G. et. al . Nature 428: 37-73, 2004; Venter, JC et al . , Science 304: 66-74, 2004). However, the existing vector system used to build such a metagenome library has a problem that is not suitable for gene expression as a vector mainly for cloning for simple purposes. In other words, when expressing a foreign gene in a heterologous expression host, various conditions such as a transcriptional regulator including a promoter, a ribosome binding site, a translational regulator such as an initiation codon, and a distance between the elements must be satisfied. It must match the host's gene transcription and translation system.

On the other hand, in order to secure a useful gene source derived from eukaryotes, a lot of methods are used to isolate the whole mRNA from the organism, convert it to cDNA, and then clone it into a vector. However, in this case, in order to express the gene in the expression host, a variety of transcriptional regulators including a promoter linked to the gene to be expressed as described above, a ribosome binding site, a translational regulator such as an initiation codon, and a distance between the elements Since conditions must be satisfied, there is a difficulty in that additional molecular biological manipulations are required to amplify the gene sequence to be expressed one by one using PCR or the like to clone it in the correct position.

Meanwhile, cell-free protein synthesis (in vitro transcription / translation system) may be used as a means for expressing foreign genes. Cell-free protein synthesis technology enables the synthesis of proteins in vitro without cell culture. For example, TNT Quick coupled Transcription translation system (Promega) system using T7 promoter. Such a system has the advantage that the peptide can be synthesized within a few hours. In addition, it is possible to prepare peptides that are degraded by proteolytic enzymes produced in the host cell and peptides that are toxic to the host cell, so that peptide derivatives or mRNAs, ribosomes and peptides that do not occur naturally do not occur. It is also possible to prepare composed complexes (Mendel D. et. al . , Annu Rev Biophys Biomol Struct. 24: 435-62, 1995). Therefore, such a system is not only easily applicable to the study of various enzymes, hormones, antibodies and the like, but also useful for molecular diagnosis, development of new medicines and catalysts, and the like. However, in order to utilize a cell-free protein synthesis system, additional molecular biological manipulations are essential to meet all of the gene expression conditions as described above. There is a disadvantage that can not.

Therefore, when the inventors want to search for useful genes through protein expression from a large number of gene sources such as metagenome, genome, or cDNA library, the transcription or translation elements contained on the foreign DNA fragment to be cloned are the gene of the host. As a result of efforts to solve the problems of the existing expression system that is not well expressed when it does not match the expression system, the most powerful T7 RNA polymerase-based T7 promoter utilization system among the E. coli expression systems currently used. In this study, a novel expression vector system was constructed to dramatically increase the expression of DNA fragments to be inserted.

Specifically, we translated the T7 promoter with lactose operator and three-frame Simultaneous Translation (TFST) constructs (three ribosomal binding sites (RBS) and different frames). A fragment comprising the three initiating codons (ATG) to which the initiation is linked is introduced into the vector so that the cloned DNA fragment is transcribed into one transcription unit (mRNA) and three frames of polypeptide A three-frame forced expression vector pTFSX that can be translated at the same time was constructed. In addition, two gene coercion expression cassettes linked to a T7 promoter, a lactose operator, and a gene three-frame co-translation construct were introduced into the vector by arranging bidirectional gene coercion cassettes arranged so as to face each other around the transcription terminator. DNA fragments cloned between two gene expression cassettes are transcribed into one transcription unit each using each of the expression genes, from which three frames of polypeptides can be translated, resulting in a total of six frames of polypeptides. A frame forced expression vector pSFSX was constructed. To verify this coexpression vector system, a green fluorescent protein gene (GFPuv) free of self-initiation codons was constructed and cloned into the frame of the coexpression cassette, and each recombinant expression vector obtained therefrom was transformed into a strain. It was confirmed that the green fluorescent protein was expressed at high levels. Through this, a novel expression vector of the present invention is transcribed into one transcription unit, respectively, in one or two directions of the cloned DNA, thereby forcibly expressing the expression of the active protein by forcibly expressing all six frames with three frames each. The present invention has been accomplished by revealing that it can be used to efficiently screen useful genes from them when applied to the construction of metagenome libraries, genomic libraries or cDNA libraries or in vitro transcription / translation systems.

An object of the present invention is to provide a novel three-frame simultaneous translation (TFST) construct, a promoter and the TFST construct to search for any gene source from the metagenome, genome, or cDNA. It is to provide a gene-forced expression vector comprising one or two or more linked gene expression cassette and a transformant to which the vector is introduced.

Another object of the present invention is to provide a method for screening any gene from metagenome using the vector according to the present invention.

In order to achieve the above object, the present invention comprises a promoter, lactose operator and three-frame Simultaneous Translation (TFST) constructs, The linked DNA fragment is transcribed into one transcription unit, from which again a gene forced expression cassette is provided in which three frames of polypeptide are simultaneously translated.

In addition,

The first gene forced expression cassette introduced forward and including promoter, lactose operator and TFST construct;

rrnB transcription terminator; And

A second gene coexpression cassette, including a promoter, a lactose operator and a TFST construct, introduced backwards of the first gene coexpression cassette,

Linking DNA fragments in place of the rrnB transcription terminator between the two gene expression cassettes, the linked DNA fragments are transcribed into one transcription unit from the bidirectional promoter, and three frames of polypeptides are bidirectionally translated from them. A bidirectional gene force expression cassette is provided in which a total of six frames of polypeptide are translated simultaneously.

In addition, the present invention provides a recombinant expression vector comprising at least one of said gene forced expression cassette or bidirectional gene forced expression cassette.

The present invention also provides a transformant in which the recombinant expression vector is introduced into a host cell.

In addition, the present invention provides a method for producing a metagenomic gene library comprising the following steps:

1) cleaving the metagenome-derived DNA with a restriction enzyme; And

2) inserting the cleaved DNA of step 1) into the expression vector according to the present invention.

In addition, the present invention provides a metagenome-derived active gene screening method comprising the following steps:

1) separating DNA from the metagenome;

2) inserting the DNA of step 1) into an expression vector according to the present invention;

3) preparing a transformant by introducing the expression vector of step 2) into a host cell; And

4) culturing the transformant of step 3) to verify protein expression.

The novel expression vector of the present invention employs a gene expression cassette in which a simple and efficient gene three-frame co-translation construct consisting of three RBS-ATGs is linked to a powerful T7 promoter, which is complex and cloned. Compared to the existing analogous expression system with the problem that protein expression is observed only in certain frames of, it is improved to show high expression in all three frames at the same time. In addition, a two-way gene expression cassette was developed by arranging two sets of gene expression cassettes to face each other so that six frames of polypeptide could be translated and expressed using one vector.

Therefore, the novel vector of the present invention not only significantly increases the possibility of expressing an active protein from a metagenome-derived gene source or the like, but is not limited to a metagenomic-derived gene, and may be a genomic library or a cDNA library. It can also be effectively used for the expression of the derived gene. In addition, the novel expression vector of the present invention can be used as a useful tool in an in vitro transcription / translation system.

Figure 1 shows three ribosome binding sites (RBS) in order to induce the cloned foreign DNA fragments to be transcribed into one transcription unit using the T7 promoter and to translate the polypeptides simultaneously into three frames. ) And a three-frame coexpression construct pTFSX for Escherichia coli containing a three-frame co-translation construct, a lactose operator (lacO), and an ampicillin antibiotic resistance gene containing three initiation codons (ATG) Schematic diagram:
ori: origin of replication;
lacO: lactose operator;
lacI: lactose repressor;
Amp: ampicillin antibiotic resistance gene;
MCS: multiple cloning site;
P _T7 : T7 promoter;
TFST: gene 3-frame co-translation construct; And
f1 ori: The origin of f1 replication.
FIG. 2 is a gene 3-frame for inducing foreign DNA fragments cloned in the novel expression vector pTFSX of the present invention to be transcribed into one transcription unit using a T7 promoter and to simultaneously translate the polypeptide into three frames. Schematic representation of the sequential arrangement of the ribosomal binding site (RBS) and initiation codon (ATG) of the co-translation construct:
T7 promoter: T7 Promoter sequence;
lacO: lactose operator;
RBS: ribosomal binding site;
ATG: start codon; And
MCS: multiple cloning site.
3 shows a gene and a T7 promoter for inducing foreign DNA fragments cloned in the novel expression vector pTFSX of the present invention to be transcribed into one transcription unit using a T7 promoter and to simultaneously translate the polypeptide into three frames. Specify the sequential arrangement of the ribosomal binding site (RBS) and initiation codon (ATG) of the three-frame co-translation construct, the multiple cloning site (MCS), and the first translation stop codon for each frame located after the multicloning site. Here is a pictorial representation of the sequence:
T7 promoter: T7 promoter;
lacO: lactose operator;
RBS: ribosomal binding site;
IC: translation initiation codon;
SC: translation stop codon;
Bam H I: Bam H I restriction enzyme recognition site;
Eco R I: Eco R I restriction enzyme recognition site;
Sac I: Sac I restriction enzyme recognition site;
Hind III: Hind III restriction enzyme recognition site;
Eag I / Not I: Eag I / Not I restriction enzyme recognition site; And
Xho I: Xho I restriction enzyme recognition site.
4 is a recombinant expression obtained by amplifying fragments so that each of the green fluorescence protein (GFPuv) genes from which the self-initiation codon was removed can be translated into three frames, and then cloned into the novel expression vector pTFSX of the present invention. A schematic representation of the vector, pTFSX-GFP1, pTFSX-GFP2 and pTFSX-GFP3 vectors.
FIG. 5 shows the amino acid sequence of the polypeptide in all cases that can be translated through three initiation codons (ATG) in the pTFSX-GFP1, pTFSX-GFP2 and pTFSX-GFP3 vectors, respectively, up to the first stop codon The result is.
FIG. 6 is a diagram showing the GFPuv gene sequence including the RBS, the start codon and the cloning site, and the amino acid sequence of the GFPuv protein expressed in the transformed strain of the DNA sequence of the recombinant expression vector pTFSX-GFP1 vector.
7 is a diagram showing the GFPuv gene sequence including the RBS, the start codon and the cloning site, and the amino acid sequence of the GFPuv protein expressed in the strain transformed with the vector among the DNA sequences of the recombinant expression vector pTFSX-GFP2 vector.
FIG. 8 is a diagram showing the GFPuv gene sequence including the RBS, the start codon, and the cloning site, and the amino acid sequence of the GFPuv protein expressed in the transformed strain of the DNA sequence of the recombinant expression vector pTFSX-GFP3 vector.
Figure 9 shows the traits recovered from the culture medium before and after induction of expression by adding IPTG to the transformed strain culture in order to confirm the expression of GFPuv in strains transformed with pTFSX-GFP1, pTFSX-GFP2 and pTFSX-GFP3 vectors After the electrophoresis of the protein obtained by crushing the transformed strain cells with 12% SDS-PAGE, and stained with Coomassie brilliant blue (coomassie brilliant blue R-250) is shown.
10 is a fluorescence of the culture medium before and after induction of expression by adding IPTG to the transformed strain culture in order to confirm the expression of GFPuv in strains transformed with pTFSX-GFP1, pTFSX-GFP2 and pTFSX-GFP3 vectors The fluorescence level is measured using a spectrometer and the figure is shown in a table.
Figure 11 shows the traits recovered from the culture medium before and after induction of expression by adding IPTG to the transformed strain culture in order to confirm the expression of GFPuv in the strains transformed with pTFSX-GFP1, pTFSX-GFP2 and pTFSX-GFP3 vectors The protein obtained by crushing the transformed strain cells was subjected to electrophoresis with 12% SDS-PAGE, followed by Western blot, and the result is shown.
FIG. 12 shows the T7 promoter, lactose, in order to induce the cloned foreign DNA fragments to be transcribed into one transcription unit each using the T7 promoter in both directions and to simultaneously translate the polypeptide into three frames each. Two gene coexpression cassettes, in which the Oz operator and three ribosome binding sites (RBSs) and the gene three-frame co-translation constructs containing three initiation codons (ATGs) are sequentially linked, provide stability of the cloning vector. Schematic representation of a six-frame coexpression vector pSFSX for Escherichia coli containing a bidirectional gene coexpression cassette arranged to face each other around a transcription terminator (TrrnB) introduced to maintain, and an ampicillin antibiotic resistance gene. Picture.
FIG. 13 shows that foreign DNA fragments cloned in the novel expression vector pSFSX of the present invention are transcribed into one transcription unit each in both directions using the T7 promoter and from which the polypeptides are simultaneously translated into three frames each. Schematic representation of the sequential arrangement of the ribosomal binding site (RBS) and initiation codon (ATG) of the bidirectional gene expression cassette:
TrrnB: rrnB transcriptional terminator.
FIG. 14 shows that foreign DNA fragments cloned in the novel expression vector pSFSX of the present invention are transcribed into one transcription unit each using a T7 promoter in both directions and from which the polypeptides are simultaneously translated into three frames each. Sequential arrangement of the ribosomal binding site (RBS) and initiation codon (ATG) of the bidirectional gene expression cassette and the Bam HI restriction enzyme cloning site are shown in the specific sequence.
FIG. 15 shows amplification of fragments so that the green fluorescence protein (GFPuv) gene from which the self-initiation codon has been removed can be translated into three frames, and then cloned into the novel expression vector pSFSX of the present invention. Recombinant expression vectors, pSFSX-5-GFP1, pSFSX-5-GFP2, pSFSX-5-GFP3, pSFSX-3-GFP1, pSFSX-3-GFP2 and pSFSX-3-GFP3 vectors are shown in the diagram. The arrow below the green fluorescent protein gene indicates the direction of green fluorescent protein gene expression by each vector.
FIG. 16 shows GFPuv gene sequences including RBS, initiation codon and cloning site, and GFPuv protein expressed in strains transformed with the vector among DNA sequences of recombinant expression vectors pSFSX-5-GFP1 and pSFSX-3-GFP1 vectors. Figure shows the amino acid sequence.
Figure 17 shows the GFPuv gene sequence including the RBS, the start codon and the cloning site in the DNA sequences of the recombinant expression vectors pSFSX-5-GFP2 and pSFSX-3-GFP2 vectors, and the GFPuv protein expressed in the strain transformed with the vector. Figure shows the amino acid sequence.
FIG. 18 shows GFPuv gene sequences including RBS, initiation codon and cloning site, and GFPuv protein expressed in strains transformed with the vector among DNA sequences of recombinant expression vectors pSFSX-5-GFP3 and pSFSX-3-GFP3 vectors. Figure shows the amino acid sequence.
19 shows the expression of GFPuv in strains transformed with pSFSX-5-GFP1, pSFSX-5-GFP2, pSFSX-5-GFP3, pSFSX-3-GFP1, pSFSX-3-GFP2 and pSFSX-3-GFP3 vectors. In order to determine the degree of fluorescence using a fluorescence spectrometer, the culture medium before and after induction of expression by adding IPTG to the transformed strain culture medium was measured and the figure is shown in a table.
20 shows the expression of GFPuv in strains transformed with pSFSX-5-GFP1, pSFSX-5-GFP2, pSFSX-5-GFP3, pSFSX-3-GFP1, pSFSX-3-GFP2 and pSFSX-3-GFP3 vectors. To this end, the protein obtained by crushing the transformed strain cells recovered from the culture medium before and after inducing expression by adding IPTG to the transformed strain culture was subjected to electrophoresis with 12% SDS-PAGE, followed by Western blot. ) And the result.

Hereinafter, the present invention will be described in detail.

The present invention comprises a T7 promoter and a three-frame Simultaneous Translation (TFST) construct [three ribosomal binding sites (RBS) and three start codons (ATG) initiated in different frames. Fragment] linked gene 3 frame forced expression cassette to the vector, the gene 3 frame forced expression vector which can be cloned DNA fragment to be transcribed into one transcription unit (mRNA) from which three frames of polypeptide can be translated simultaneously pTFSX was produced. In addition, a gene 6-frame coexpression vector pSFSX, in which two gene 3-frame coercion expression cassettes were opposed to each other based on a translation terminator (TrrnB) sequence, was produced. The green fluorescent protein gene (GFPuv) from which the self-initiation codon was removed from the cassette was cloned into three frames, and each recombinant expression vector obtained from them was expressed at high levels in each of the transformed strains. Confirmed.

The present invention includes a promoter, a lactose operator, and a Genetic Three-Frame Simultaneous Translation (TFST) construct, wherein when the DNA fragments are linked, the linked DNA fragment is linked to one transcription unit. To a gene 3 frame forced expression cassette from which 3 frames of polypeptide are simultaneously translated.

The promoter is preferably a T7 promoter, but is not limited thereto. Any promoter capable of inducing the expression of a foreign gene in E. coli may be used.

The promoter, lactose operator and gene 3-frame co-translation constructs are preferably included sequentially, but are not limited thereto.

The gene 3-frame co-translation construct may include a promoter, a lactose operator, a ribosomal binding site (RBS), a ribosomal binding site (RBS), an initiation codon (ATG), a ribosome binding site (ribosome binding site, RBS), start codon (ATG), start codon (ATG) is preferably operably linked sequentially, but is not limited thereto.

The gene forcing expression cassette preferably has the structure described in FIG. 2, but is not limited thereto.

The gene 3-frame co-translation construct is preferably composed of the nucleotide sequence of SEQ ID NO: 14, but is not limited thereto.

The ribosomal binding site (RBS) is preferably "aggaga" which is the nucleotide sequence of SEQ ID NO: 7, but is not limited thereto.

In a specific embodiment of the invention, a gene coexpression cassette consisting of a T7 promoter, a lactose operator, and a gene three-frame co-translation construct comprising three ribosome binding sites (RBS) and three initiation codons (ATG) And a foreign DNA fragment to be cloned was transcribed into one transcription unit (mRNA), from which a three-frame forced expression vector pTFSX was constructed in which three frames of polypeptide could be simultaneously translated (FIGS. 1 to 3). Reference).

In addition,

The first gene forced expression cassette introduced forward and including promoter, lactose operator and TFST construct;

rrnB transcription terminator; And

A second gene coexpression cassette, including a promoter, a lactose operator and a TFST construct, introduced backwards of the first gene coexpression cassette,

Linking DNA fragments in place of the rrnB transcription terminator between the two gene expression cassettes, the linked DNA fragments are transcribed into one transcription unit from the bidirectional promoter, and three frames of polypeptides are bidirectionally translated from them. A bidirectional gene force expression cassette is provided in which a total of six frames of polypeptide are translated simultaneously.

The promoter is preferably a T7 promoter, but is not limited thereto. Any promoter capable of inducing the expression of a foreign gene in E. coli may be used.

The promoter, lactose operator and gene 3-frame co-translation constructs are preferably included sequentially, but are not limited thereto.

The TFST construct of the first gene coexpression cassette of the bidirectional gene coexpression cassette is a sequentially operably linked ribosome binding site, ribosomal binding site, initiation codon, ribosomal binding site, initiation codon and initiation codon, second gene The TFST construct of the coexpression cassette is preferably a start codon, a start codon, a ribosomal binding site, a start codon, a ribosomal binding site, and a ribosomal binding site sequentially linked to be operably linked in the reverse direction of the first TFST construct. It is not limited to this.

The bidirectional gene forced expression cassette preferably has the structure described in FIG. 13, but is not limited thereto.

The TFSX construct of the first gene expression cassette is preferably composed of the nucleotide sequence of SEQ ID NO: 14, the TFSX construct of the second expression cassette is preferably composed of the nucleotide sequence of SEQ ID NO: 24, but is not limited thereto.

The ribosomal binding site (RBS) is preferably "aggaga" which is the nucleotide sequence of SEQ ID NO: 7, but is not limited thereto.

The rrnB transcription terminator is preferably composed of the nucleotide sequence of SEQ ID NO: 23, but is not limited thereto.

In a specific embodiment of the present invention, two gene-expression cassettes sequentially linked by a TFST comprising a T7 promoter, a lactose operator and three ribosomal binding sites (RBS) and three initiation codons (ATG) are centered on the rrnB transcription terminator. And foreign DNA fragments which are introduced to face each other, and which are cloned between the two gene expression cassettes instead of the rrnB transcription terminator, are transcribed from the bidirectional promoter into one transcription unit (mRNA) each From these, three frames of polypeptides were bidirectionally translated, respectively, to produce a forced expression vector pSFSX capable of simultaneously translating a total of six frames of polypeptides (see FIGS. 12 to 14).

In addition, the present invention provides a recombinant expression vector comprising at least one gene forcing expression cassette produced above.

The expression vector may be expressed by transcoding the DNA fragment to be cloned into one transcription unit and translated into three frames of polypeptide, or the DNA fragment to be cloned may be expressed by one transcription unit in each direction from each promoter. Is transcribed into, and each of three frames of polypeptides is translated so that a total of six frames of polypeptides can be translated and expressed, but is not limited thereto.

The recombinant expression vector preferably comprises one or more gene-forced expression cassettes, more preferably one to three, most preferably one to two, but is not limited thereto.

The present invention also provides a transformant in which the recombinant expression vector is introduced into a host cell.

The host cell is preferably E. coli , but is not limited thereto. Any host cell capable of introducing a recombinant expression vector may be used.

In a specific embodiment of the present invention, two gene-expression cassettes sequentially linked by a TFST comprising a T7 promoter, a lactose operator and three ribosomal binding sites (RBS) and three initiation codons (ATG) are centered on the rrnB transcription terminator. And foreign DNA fragments which are introduced to face each other, and which are cloned between the two gene expression cassettes instead of the rrnB transcription terminator, are transcribed from the bidirectional promoter into one transcription unit (mRNA) each From each of these, three frames of polypeptides were bidirectionally translated to produce a forced expression vector pSFSX capable of simultaneously translating a total of six frames of polypeptides.

Subsequently, each recombinant expression vector was constructed by cloning the green fluorescent protein gene from which the self-initiation codon was removed (see FIGS. 4 to 8, and FIGS. 15 to 18), and then the recombinant expression vector was transformed. Strains were selected, and it was confirmed that the GFPuv protein was expressed at high levels in all of the selected strains (see FIGS. 9, 11, and 20), and exhibited significantly high fluorescence (see FIGS. 10 and 19).

This allows DNA fragments to which the novel expression vectors of the invention are cloned can be transcribed into one transcription unit and translated into three frames of polypeptide, or the DNA fragments cloned from each promoter It can be seen that one frame is transcribed in both directions, from which three frames of polypeptide are translated, thereby six frames of polypeptide can be translated and expressed.

In encoding DNA into proteins, the base constituting the DNA consists of three units of one codon, and one codon encodes one amino acid. The gene 3-frame co-translation construct included in the vector of the present invention is composed of three ribosomal binding sites (RBS) and three initiation codons (ATG), which are translated differently by one base unit, thereby converting the polypeptide into three frames. It can be encrypted.

Therefore, when the codon is changed by one base unit, it is possible to translate three polypeptides of all cases encoded in the same direction, and when using the novel expression vector of the present invention, the cloned DNA is different in the forward and reverse directions. Since it can be translated into three frames of polypeptide, foreign genes can be forcibly expressed and used to search for active protein genes from a large amount of genomic resources such as metagenome, genome, or cDNA library.

The novel vector of the present invention consists of a simple arrangement of three RBS-ATGs, which allows simple expression of the gene and expression of the protein in all three frames, and powerful expression of all the expressed proteins in the three frames. As a system, since a gene can be transcribed by a single base unit through a specific arrangement in a vector, it can be usefully used to search for an active gene source of a metagenome gene.

In addition, the present invention provides a method for producing a metagenome-derived gene library comprising the following steps:

1) cleaving the metagenome-derived DNA with a restriction enzyme; And

2) inserting the cleaved DNA of step 1) into the expression vector of the present invention.

The cleavage of step 1) preferably further includes a step of partially cleaving with a Sau 3A1 restriction enzyme or a step of making a blunt end after mechanical cleavage, but is not limited thereto.

The present invention also provides a method for screening an active gene derived from a metagenome, comprising the following steps:

1) separating DNA from the metagenome;

2) inserting the DNA of step 1) into the expression vector of the present invention;

3) preparing a transformant by introducing the expression vector of step 2) into a host cell; And

4) culturing the transformant of step 3) to verify protein expression.

The protein expression verification of step 4) is preferably performed by any one method selected from the group consisting of immunofluorescence, enzyme immunoassay (ELISA), Western blot and RT-PCR, but is not limited thereto.

In specific embodiments of the invention, one or two gene coexpression cassettes are sequentially linked to a TFST comprising a T7 promoter, a lactose operator and three ribosomal binding sites (RBS) and three initiation codons (ATG) Each recombinant expression vector was produced by constructing a forced expression vector and cloning the green fluorescent protein gene from which the self-initiation codon was removed. Then, it was confirmed that all of the green fluorescent proteins were expressed at high levels in the transformed strains of the recombinant expression vector. Therefore, the novel vectors of the present invention can be usefully used for the construction of metagenomes, genomes, or cDNA libraries, or for screening active genes from them. In addition, the novel expression vector of the present invention may be usefully used in an in vitro transcription / translation system, which is a cell-free protein synthesis system.

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are only illustrative of the present invention in detail, and the content of the present invention is not limited by the examples.

< Example  1> New Expression Vectors to Induce Three Frame Expression pTFSX  making

PET-21a + (Novagen, EMD Biosciences Inc, Madison, USA) plasmid was used to construct a novel expression vector for inducing three frame expression of the present invention.

Specifically, pET-21a + (Novagen, EMD Biosciences Inc, Madison, USA) plasmid as a template, using primer pairs of SEQ ID NO: 1 and 2, using PrimeStar HS polymerase (Takara, Japan) for 10 seconds at 98 ℃ Polymer chain reaction (Polymerase Chain Reaction, PCR) was performed at 58 ° C. for 5 seconds, 72 ° C. for 5 minutes, and 30 cycles to amplify DNA fragments having a size of 5,426 bp. The amplified DNA fragment was purified using a DNA separation purification kit (Xprep PCR purification kit, JMC R & D, INC., Daejeon, South Korea) and purified by restriction enzyme Bam HI (TaKaRa Bio Inc., Shiga, Japan). E. coli DH5α was transformed by self-ligation. Thereafter, plasmid DNA was isolated from the recombinant E. coli transformed, and the isolated DNA was sequenced (sequencing, Genotech, Daejeon, South Korea) to confirm its nucleotide sequence.

As a result, it was confirmed that an expression vector capable of transcribing the DNA fragment to be cloned into one transcription unit and translating three frames of polypeptides simultaneously was named pTFSX (FIGS. 1 to 3).

< Example  2> pTFSX  In my vector Green fluorescence  protein( GFPuv A) gene Cloning

In order to confirm whether the novel expression vector pTFSX produced in the present invention can be transcribed into one transcription unit and three frames of the polypeptide can be translated from each other, a green fluorescent protein ( Green fluorescence protein (GFP) gene was cloned.

Specifically, cloning of the green fluorescent protein (GFPuv) gene is based on pGFPuv (Clontech Laboratories, Inc., Palo Alto, Calif. USA) as a template, GFP 1 forward primer of SEQ ID NO: 3, GFP 2 forward primer of SEQ ID NO: 4 Alternatively, GFP 3 forward primer of SEQ ID NO: 5 and GFP reverse primer of SEQ ID NO: 6 are combined in a pair of respective forward primers and reverse primers, using PrimeStar HS polymerase (Takara, Japan) at 98 ° C for 10 seconds, 58 ° C. PCR was performed at 5 seconds, 72 ° C., 30 seconds, and 30 cycles. The GFP 1, GFP 2, and GFP 3 gene fragments, which can be removed by self-initiation codons obtained through the PCR amplification and translated into three different frames, were subjected to PCR product purification kit (Xprep PCR purification kit, JMC R & D, INC., After pure purification using Daejeon, South Korea) and processing the restriction enzyme Bgl II (TaKaRa Bio Inc., Shiga, Japan), it was prepared by insert (insert). At this time, the expression vector pTFSX prepared in Example 1 was digested with restriction enzyme Bam HI (TaKaRa Bio Inc., Shiga, Japan) and prepared by treating with dephosphorase, and the prepared insert and lye were prepared. Ligation was performed. Thereafter, the ligated mixture was transformed into E. coli DH5α, plasmid DNA was isolated from the resulting recombinant transformants, and DNA fragment size confirmation and restriction analysis were performed through restriction sequencing and DNA sequencing.

As a result, it was confirmed that three gene fragments of GFP 1, GFP 2 and GFP 3 were cloned into the expression vector pTFSX vector, and the resulting recombinant expression vectors were named pTFSX-GFP1, pTFSX-GFP2 and pTFSX-GFP3 (Fig. 4).

At this time, as shown in Figure 5, each of the produced recombinant vector has three start codons, but if you check the number of all cases that can be translated in the three start codons, when the GFPuv gene is normally translated (Red arrow) indicates that the amino acid sequence of the polypeptide to be translated coincides with the GFPuv protein and there is no stop codon (*) in the middle of the sequence of the corresponding translation frame, and in other frames, the amino acid of the polypeptide to be translated It was confirmed that the sequence was completely different from the GFPuv protein and also there was a stop codon in the middle, which could not be translated into a normal GFPuv protein (FIG. 5).

< Example  3> green fluorescent protein ( GFPuv ) Expression Detection in Three Frames Using Genes

Experiments were performed to determine whether GFPuv protein was expressed in a recombinant vector cloned with the green fluorescent protein (GFPuv) gene in pTFSX, a novel expression vector of the present invention.

<3-1> Culture of Recombinant Transformant Strain

PTFSX-GFP1, pTFSX-GFP2, and pTFSX-GFP3 plasmids prepared in <Example 2> were transformed to E. coli BLR (DE3), a RecA disrupted strain, by electroporation to prepare a recombinant transformant, and this was Luria. Pre-incubated at 37 ° C. with Bertani (LB) medium (Tryptone 10 g / L, Yeast extract 5 g / L, Sodium chloride 10 g / L). Thereafter, the preculture was inoculated again in LB medium at a volume ratio of 100%, incubated at 37 ° C. until absorbance 600 nm 0.6, and then isopropyl-beta to induce the expression of green fluorescent protein (GFPuv). -Thiogalactopyranoside (isopropyl-β-D-thiogalactopyranoside, IPTG, Duchefa, the Netherlands) was added to a final 0.5 mM, and further incubated at 16 ° C for 24 hours, followed by centrifugation of the culture to recover the cells. .

<3-2> In recombinant transformants GFPuv Confirmation of expression of

SDS-PAGE and Western blot analysis of the recombinant transformants confirmed the expression level and pattern of GFPuv.

Specifically, SDS-PAGE analysis was performed by crushing the cells recovered in the culture medium of <Example 3-1> and separating the obtained intracellular protein using 12% SDS-PAGE, Coomassie brilliant blue R (coomassie brilliant blue R -250).

Western blot analysis was also performed by separating proteins via 10% SDS-PAGE and transferring the proteins to PVDF membranes using an electrical method (40 minutes at 80V). The protein was transferred to the PVDF membrane treated with TBST blocking buffer containing 5% skim milk for 1 hour, monoclonal GFPuv antibody (Santa cruze) as a primary antibody using TBST buffer 1/1 Diluted at a rate of 1000, treated for 1 hour, and then washed three times at 10 minute intervals using TBST, and the secondary antibody ( Anti-mouse HRP-conjugated secondary antibody, Sigma-Aldrich) at a rate of 1/2000 , St. Louis, MO, USA), followed by three washes at 10 minute intervals using TBST, followed by detection of the target protein from the PVDF membrane using ECL solution (GE Healthcare).

As a result, as shown in FIGS. 9 and 11, about 27 kDa fluorescent protein was expressed only when the expression of GFPuv was induced by IPTG in the recombinant strain transformed with the pTFSX-GFP1, pTFSX-GFP2, and pTFSX-GFP3 vectors. (FIGS. 9 and 11), through which, it was confirmed that GFPuv was expressed in a frame in each recombinant vector transformed strain.

<3-3> fluorescence spectrometer ( 여광체 분광계 ) GFPuv Confirmation of expression of

In order to confirm the expression of GFPuv using the fluorescence spectrometer in the recombinant transformant, the cells recovered in the culture medium of <Example 3-1> were crushed, and the obtained intracellular protein was used as a sample, a fluorescence spectrometer. , Shimadzu RF5301PC, Japan) was used to measure the fluorescence of each sample at an emission wavelength of 395 nm and an emission wavelength of 509 nm.

As a result, as shown in FIG. 10, only when the expression of GFPuv was induced by IPTG in each recombinant transformant, significantly high fluorescence was observed (FIG. 10), whereby each recombinant vector was transformed. It was confirmed that GFPuv was expressed in a frame according to the strain.

< Example  4> New Expression Vectors to Induce Six Frame Expression pSTFX  making

A novel expression vector was constructed capable of inducing the six frame expressions of the invention.

First, a rrnB transcription terminator was inserted to maintain the stability of the TFSX gene expression cassette in the pTFSX vector prepared in Example 1, specifically, a pTrc99a vector (Amann, E. et al, Gene). 69 (2): 301-315, 1988) as a template, using SEQ ID NOs: 15 and 18 primer pairs, and PrimeStar HS polymerase using 98 ° C 10 seconds, 58 ° C 5 seconds, 72 ° C 30 seconds , Polymerization Chain Reaction (PCR) was carried out under the conditions of 30 cycles, amplified 407 bp TrrnB DNA fragment and purified by restriction enzyme Bam HI, and then inserted into the insert. (insert) was cloned into the Bam HI cleavage site of pTFSX and named it pTFSX-TrrnB.

Then, using the pTFSX-TrrnB plasmid as a template, using SEQ ID NO: 16 and 22 primer pairs and SEQ ID NO: 20 and 21 primer pairs, respectively, 98 ℃ 10 seconds, 58 ℃ 5 seconds, 72 ℃ 6 minutes, 30 cycles ( cycles) and PCR under the conditions of 98 ° C. 10 sec, 58 ° C. 5 sec, 72 ° C. 20 sec, and 30 cycles to obtain amplified products of 5,675 bp and 139 bp, which were amplified through the pure purification process. After the ligation was cut with restriction enzymes Eco RI and Sma I and ligation was performed. Since there are three Bam HI cleavage sites in the constructed vector, after treatment with the restriction enzyme Bam HI, the amplified rrnB terminator was inserted into the Bam HI cleavage site to prepare a 681bp SFSX cassette. It was.

Then, it was again treated with restriction enzyme Bgl II, and inserted into the Bam HI and Bgl II cleavage sites of the pET-21a + plasmid, which was transformed into E. coli DH5α, and the plasmid DNA was isolated from E. coli transformed with the recombinant plasmid. The separated DNA was sequenced (sequencing, Genotech, Daejeon, Korea) to confirm the base sequence.

At this time, all the amplified DNA fragments were purified using a DNA purification kit (Xprep PCR purification kit, JMC R & D, INC., Daejeon, South Korea), and transformed into E. coli DH5α.

As a result, the cloned DNA fragment was transcribed into one transcription unit from the bidirectional promoter, and 3 frames of polypeptide were translated from them, thereby confirming that an expression vector capable of simultaneously translating a total of 6 frames of polypeptides was produced. It was named pSFSX.

< Example  5> pSFSX  In my vector Green fluorescence  protein( GFPuv A) gene Cloning

In order to confirm whether or not a total of six frames of polypeptides can be translated by transcribing a single transcription unit from a bidirectional promoter and translating three frames of polypeptides using the novel expression vector pSFSX produced in the present invention, First, the green fluorescence protein (GFP) gene was cloned into the prepared pSFSX vector.

First, in order to introduce the rrnB transcription terminator into the pGFPuv (ClontechLaboratories, Inc., Palo Alto, Calif. USA) plasmid, pTrc99a was used as a template, and SEQ ID NOs: 17 and 19 primer pairs were used, and PrimeStar HS polymerase. A polymerase chain reaction (PCR) was performed under conditions of 98 ° C. 10 seconds, 58 ° C. 5 seconds, 72 ° C. 30 seconds, and 30 cycles to amplify a DNA fragment having a size of 407 bp, After pure purification, it was digested with restriction enzyme Eco RI and cloned into Eco RI cleavage site of pGFPuv.

Then, as a template, a GFP 1 forward primer of SEQ ID NO: 3, a GFP 2 forward primer of SEQ ID NO: 4, or a GFP 3 forward primer of SEQ ID NO: 5 and a rrnB transcription terminator GFP reverse primer of SEQ ID NO: 19, respectively, PCR was performed under the conditions of 98 ℃ 10 seconds, 58 ℃ 5 seconds, 72 ℃ 1 minutes, 30 cycles using PrimeStar HS polymerase (Takara, Japan) by combining a pair of the forward primer and the reverse primer.

The PCR amplification yielded GFP1-TrrnB, GFP 2-TrrnB, and GFP 3-TrrnB gene fragments, which can be self-initiated codons removed and translated into three different frames, which were then subjected to PCR product purification kit (Xprep PCR purification kit). , JMC R & D, INC., Daejeon, South Korea) was purified pure and treated with restriction enzyme Bgl II (TaKaRa Bio Inc., Shiga, Japan), it was prepared as an insert (insert).

At this time, the expression vector pSFSX prepared in Example 4 was cut with restriction enzyme Bam HI (TaKaRa Bio Inc., Shiga, Japan) and prepared by treating with dephosphorylated enzyme, and then prepared with the prepared insert (inset) and lye. Ligation was performed. Thereafter, the ligated mixture was transformed into E. coli DH5α, plasmid DNA was isolated from the resulting recombinant transformants, and DNA fragment size confirmation and restriction analysis were performed through restriction sequencing and DNA sequencing.

As a result, three gene fragments of GFP 1, GFP 2 and GFP 3 in the forward direction and three gene fragments of GFP 1, GFP 2 and GFP 3 in the forward direction were respectively cloned into the expression vector pSFSX vector, resulting in a total of six gene fragments. The cloned recombinant expression vector was then identified as pSFSX-5-GFP1, pSFSX-5-GFP2, pSFSX-5-GFP3, pSFSX-3-GFP1, pSFSX-3-GFP2 and pSFSX-3-GFP3. It was named (FIG. 15).

< Example  6> green fluorescent protein ( GFPuv Expression Detection in Six Frames Using Genes

Experiments were performed to confirm the expression of GFPuv protein in the recombinant vector cloned with the green fluorescent protein (GFPuv) gene in pSFSX, a novel expression vector of the present invention.

<6-1> Culture of Recombinant Transformant Strain

PSFSX-5-GFP1, pSFSX-5-GFP2, pSFSX-5-GFP3, pSFSX-3-GFP1, pSFSX-3-GFP2 and pSFSX-3-GFP3 plasmids prepared in Example 5 were electroporated. E. coli BLR (DE3) was transformed to produce a recombinant transformant, which was subjected to Luria-Bertani (LB) medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L) at 37 ° C. Preincubation for at least 12 hours. Thereafter, the preculture was inoculated again in LB medium at a volume ratio of 100%, incubated at 37 ° C. until absorbance of 600 nm 0.6, and then isopropyl-beta to induce the expression of green fluorescent protein (GFPuv). -Thiogalactopyranoside (isopropyl-β-D-thiogalactopyranoside, IPTG, Duchefa, the Netherlands) was added to a final 0.5 mM, and further incubated at 16 ° C for 24 hours, followed by centrifugation of the culture to recover the cells. .

<6-2> in recombinant transformants GFPuv Confirmation of expression of

In order to confirm the expression level and pattern of GFPuv in the recombinant transformants, Western blot analysis was performed as in the method of Example <3-2>.

As a result, as shown in FIG. 20, pSFSX-5-GFP1, pSFSX-5-GFP2, pSFSX-5-GFP3, pSFSX-3-GFP1, pSFSX-3-GFP2 and pSFSX-3-GFP3 vectors were transformed. It was confirmed that about 27 kDa fluorescent protein was expressed only when the expression of GFPuv was induced by IPTG in the recombinant strain (FIG. 20), thereby confirming that GFPuv was expressed in a frame in each of the transformed strains. It was.

<6-3> fluorescence spectrometer ( 여광체 분광계 ) GFPuv Confirmation of expression of

In order to confirm the expression of GFPuv using a fluorescence spectrometer in the recombinant transformant, the cells recovered from the culture medium of <Example 6-1> were disrupted and the obtained intracellular protein was used as a sample. As in the method of 3>, the fluorescence of each sample was measured.

As a result, as shown in FIG. 19, only when the expression of GFPuv was induced by IPTG in each recombinant transformant, significantly high fluorescence was observed (FIG. 19), whereby each recombinant vector was transformed. It was confirmed that GFPuv was expressed in a frame according to the strain.

As described above, the novel expression vectors of the present invention can simultaneously translate the DNA fragment to be cloned into three different frames by one base unit. Therefore, when the expression vectors are used to produce a library of metagenome, genome, and cDNA derived genes, expression and activity screening of useful unknown genes included in the library are easy. In other words, the genome library can easily search for a wide range of metagenomes, genomes, and cDNA gene sources through the production of active genome libraries that can easily express active proteins. Metabolites, etc.) or pharmaceutical materials (antibiotics, recombinant drugs) can be usefully used.

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Claims (17)

An expression cassette that includes a promoter, lactose operator (lacO), and a Genetic Three-Frame Simultaneous Translation (TFST) construct. A gene forced expression cassette, which is transcribed in a transcription unit, from which three frames of polypeptide are simultaneously translated.
The method of claim 1, wherein the TFST construct is a ribosome binding site (RBS), ribosomal binding site (RBS), initiation codon (ATG), ribosomal binding site (RBS), A gene coexpression cassette characterized in that the start codon (ATG) and the start codon (ATG) are operably linked in sequence.
The gene-forced expression cassette according to claim 1, wherein the TFST construct is composed of the nucleotide sequence of SEQ ID NO.
The first gene forced expression cassette introduced forward and including promoter, lactose operator and TFST construct;
rrnB transcription terminator; And
A second gene forced expression cassette comprising a promoter, a lactose operator and a TFST construct and introduced in reverse of the first gene forced expression cassette,
Linking the DNA fragments in place of the rrnB transcription terminator between the two gene expression cassettes, the linked DNA fragments are transcribed into one transcription unit from the bidirectional promoter, and three frames of polypeptides are bidirectionally translated from them. A bidirectional gene force expression cassette in which a total of six frames of polypeptides are translated simultaneously.
5. The method of claim 4, wherein the TFST construct of the first gene forced expression cassette of the bidirectional gene forced expression cassette comprises sequentially operably linked ribosomal binding sites, ribosomal binding sites, start codons, ribosomal binding sites, start codons, and start codons. And the TFST construct of the second gene-forced expression cassette can be operated in reverse reciprocation of the first TFST construct, with the start codon, the start codon, the ribosome binding site, the start codon, the ribosome binding site, and the ribosomal binding site sequentially linked. Gene expression cassette characterized in that the connection.
5. The gene-forced expression cassette of claim 1 or 4, wherein the promoter is a T7 promoter.
The gene-forced expression cassette according to claim 2 or 5, wherein the ribosomal binding site (RBS) is composed of the nucleotide sequence of SEQ ID NO.
5. The gene-forced expression cassette of claim 4, wherein the rrnB transcription terminator consists of the nucleotide sequence of SEQ ID NO: 23.
5. The gene-forced expression cassette of claim 4, wherein the TFST construct of the first expression cassette consists of the nucleotide sequence of SEQ ID NO: 14, and the TFST construct of the second expression cassette consists of the nucleotide sequence of SEQ ID NO: 24. .
A recombinant expression vector comprising the gene forced expression cassette of claim 1.
The method of claim 10, wherein the expression vector can be expressed by transcribing the DNA fragment to be cloned into one transcription unit and translated into three frames of polypeptide, or the DNA fragment to be cloned from each promoter. An expression vector, wherein the expression vector is transcribed into one transcription unit in each direction, and three frames of polypeptides are translated from each of them, so that a total of six frames of polypeptides can be translated and expressed.
A transformant, wherein the recombinant expression vector of claim 10 is introduced into a host cell.
The transformant of claim 12, wherein the host cell is E. coli .
1) cleaving the metagenome-derived DNA with a restriction enzyme; And
2) A method for producing a metagenome derived gene library comprising inserting the cut DNA of step 1) into the expression vector of claim 10.
The method of claim 14, wherein the cleavage of step 1) further comprises partially cleaving with a Sau 3A1 restriction enzyme.
15. The method of claim 14, wherein the cleavage of step 1) further comprises the step of making a blunt end after mechanical cleavage.
1) separating DNA from the metagenome;
2) inserting the DNA of step 1) into the expression vector of claim 10;
3) preparing a transformant by introducing the expression vector of step 2) into a host cell; And
4) culturing the transformant of step 3) to verify protein expression, metagenomic derived active gene screening method.

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