JP4838434B2 - Protein expression system using plant body - Google Patents

Description

【図面の簡単な説明】
【図１】ｐＬＤ６の模式図を示す。Ｐｒｒｎはタバコ葉緑体由来のｒｒｎプロモーターを表す。該ｒｒｎプロモーターは、配列番号２で表される塩基配列を持ち、配列番号１中の２２２６−２３６８に位置する。ａａｄＡはスペクチノマイシン耐性遺伝子を表す。該スペクチノマイシン耐性遺伝子は、配列番号３で表される塩基配列を持ち、配列番号１中の２３６８−３１７３に位置する。ＴｐｓｂＡはタバコ葉緑体由来のｐｓｂＡターミネーターを表す。該ｐｓｂＡターミネーターは配列番号４で表される塩基配列を持ち、配列番号１中の３１７５−３５６８に位置する。ＰｐｓｂＡはタバコ葉緑体由来のｐｓｂＡプロモーターを表す。該ｐｓｂＡプロモーターは配列番号５で表されるで表される塩基配列を持ち、配列番号1中の３５６９−３７０１に位置する。Ｔｒｐｓ１６はタバコ葉緑体由来のｒｐｓ１６ターミネーターを表す。該ｒｐｓ１６ターミネーターは、配列番号６で表される塩基配列を持ち、配列番号１中の３７５５−３９１３に位置する。塩基配列を記載した部位はマルチクローニング領域である。残りの部分（太線の部分）はpBluescript II SK(+)由来の遺伝子である。BglII、SphI、ClaIおよびEcoRIは制限酵素部位を表し、ＳＤはＳＤ配列（図１に示した塩基配列の５'末端から数えて１３番目から始まる５ヌクレオチドからなるセグメント）を表す。
【図２】ＧＦＰ遺伝子が発現している実施例で得られた葉緑体形質転換体を有するタバコの外観を示す。
【図３】ＧＦＰ遺伝子が発現している実施例で得られた葉緑体形質転換体を有するタバコの葉の蛍光像を示す。
【図４】実施例で得られた葉緑体形質転換体を有するタバコからタンパク質を抽出しＳＤＳ−ＰＡＧＥを行った（パネルＡ）。また、ＧＦＰ遺伝子の導入されていない野生型のタバコを用いて同様にＳＤＳ−ＰＡＧＥを行った（パネルＢ）。全可溶タンパク質量の５０％をしめるRuBisCO(rbcL+rbcS)に比較しうる量のＧＦＰの蓄積が実施例で得られたＧＦＰ遺伝子導入タバコで観察された。なお、パネルＭは分子量マーカーである。
【図５】ｐＬＤ２００の模式図を示す。ｒｂｃＬはタバコ葉緑体由来のｒｂｃＬ遺伝子を表す。ａｃｃＤはタバコ葉緑体由来のａｃｃＤ遺伝子を表す。塩基配列を記載した部位のうち５'末端から数えて５番目から２１塩基はポリリンカーである。太線の部分はｐＵＣ１９由来の遺伝子である。NotI、NheIおよびSphIは制限酵素部位を表す。
【図６】ｐＬＤ２００の構築過程を示す。
【図７】ｐＬＤ６の構築過程のうち第１過程を示す。
【図８】ｐＬＤ６の構築過程のうち第２過程を示す。
【図９】ｐＬＤ６の構築過程のうち第３過程を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a protein expression system using a plant body.
[0002]
[Prior art]
In the case of preparing a large amount of useful proteins that can be used as pharmaceuticals using genetic recombination, a system using E. coli has been widely used so far. In this case, it is considered that toxins are contaminated in the purification process of the expressed protein, and in such a case, the use of animal cells has been put to practical use. However, the cost of culturing when using animal cells is several tens of times that of E. coli, and the resulting product (protein) is expensive. In this respect, since plants grow by photosynthesis, the input of energy is small, and the product (protein) can be inexpensive. In fact, when the product unit price is compared as a host for obtaining a genetically modified product, it is estimated that animal cells, Escherichia coli, and plants will be cheaper in this order. For this reason, development of the mass protein expression system of the useful protein using the plant body was eagerly desired.
[0003]
There are about 100 chloroplasts per cell in adult plants, and there are 100 copies of chloroplast genome genes per chloroplast (Bendich, AJ, Why do chloroplasts and mitochondria contain so many copies). of their genome?Bioassays,6, 279-282 (1987)). This means that if one copy of a foreign gene is inserted into the chloroplast genome, there will be 10,000 copies per cell in the transformant, and high expression can be expected from the large number of copies (Maliga, P., Towards plastid transformation in flowering plants.Trends Biotechnol.11, 101-107 (1993)). Furthermore, since gene introduction into chloroplasts utilizes homologous recombination, the position effect seen at the time of insertion into the nucleus does not occur, and stable gene expression is performed. In addition, since chloroplasts are maternally inherited, gene transfer into chloroplasts has many advantages, such as prevention of scattering into the environment via pollen of the introduced gene (see the above document).
[0004]
Overexpression of foreign proteins has been achieved using chloroplast transformation techniquesBacillusToxins produced by McBride, K.E., Svab, Z., Schaaf, D.J., Hogan, P.S., Stalker, D.M. and Maliga, P. Amplification of a chimericBacillus gene in chloroplasts leads to an extraordinary level of an insecticidal protein in tobacco.Bio / technol.,13, 362-365, or Kota, M., Daniell, H., Varma, S., Garczynski, S.F., Gould, F., and Moar, W.J., Overexpression of theBacillus thuringiensis (Bt) Cry2Aa2 protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects.Proc. Natl. Acad. Sci. USA, 96, 1840-1845 (1999)), herbicides (Daniell, H., Datta, R., Varma, S., Gray, S., and Lee, S.-B., Containment of herbicide resistance through genetic engineering of the chloroplast genome.Nature Biotechnol.16, 345-348. (1998)), but in these cases, the total expression of the total soluble fraction was determined because polycistonic gene expression with the aadA gene for selection of transformants was targeted. It was about 6-7% of the minute.
[0005]
[Problems to be solved by the invention]
An object of this invention is to provide the vector for protein expression using a plant body.
The present invention also has an expression vector capable of highly expressing a target protein in chloroplasts, a transformed chloroplast transformed with the expression vector, and further, the transformed chloroplast. The purpose is to provide plants. Furthermore, it aims at providing a vector useful for said expression vector preparation.
[0006]
[Means for Solving the Problems]
As a result of intensive studies on a protein expression system using a plant body, particularly a protein expression system using a chloroplast, the present inventors introduce a psbA promoter derived from tobacco chloroplast upstream of a gene encoding the protein. Thus, an unexpected finding that the protein encoded by the introduced gene can be highly expressed in tobacco chloroplasts was obtained. Furthermore, as a result of studying to increase the expression level of the protein encoded by the introduced gene, the expression level of the protein encoded by the introduced gene is increased by placing a ribosome binding site upstream of the translation start point of the gene encoding the protein. We obtained an unexpected finding that it would increase further.
The present inventors have further studied the protein expression system in the plant body and completed the present invention.
[0007]
That is, the present invention
(1) (a) a plurality of restriction enzyme sites, an insertion site into which a gene encoding a protein sandwiched between two restriction enzyme sites can be inserted, a multicloning region having a ribosome binding site upstream of the insertion site, and multicloning A pre-construction gene group having a promoter upstream of the region and a terminator downstream of the multi-cloning region; (b) a gene for identifying a gene recombinant upstream thereof; a promoter upstream thereof; and a terminator downstream thereof. A vector having a discriminating gene group,
(2) The vector according to (1) above, wherein the multicloning region of the pre-constructed gene group has a ribosome binding site 7 to 11 bases upstream of the translation start point of the gene encoding the protein,
(3) The vector according to (1) or (2) above, wherein the ribosome binding site is an SD sequence,
(4) The vector according to (1) to (3) above, wherein the multicloning region of the pre-constructed gene group is composed of the base sequence represented by SEQ ID NO: 7,
(5) The vector according to (1) to (4) above, wherein the promoter and terminator of the pre-construction gene group or / and the promoter and terminator of the identification gene group are a chloroplast-derived promoter and terminator ,
(6) The vector according to (5) above, wherein the chloroplast-derived promoter and terminator are a tobacco chloroplast-derived promoter and terminator,
(7) The vector according to (6) above, wherein the promoter derived from tobacco chloroplast is psbA promoter or rrn promoter derived from tobacco chloroplast,
(8) The vector according to (6) or (7), wherein the terminator derived from tobacco chloroplast is a psbA terminator or rps16 terminator derived from tobacco chloroplast,
(9) The vector according to (6) above, wherein the promoter of the pre-construction gene group is a psbA promoter derived from tobacco chloroplasts,
(10) The promoter of the pre-construction gene group is a psbA promoter derived from tobacco chloroplast, the terminator of the pre-construction gene group is rps16 terminator derived from tobacco chloroplast, and the promoter of the identification gene group is derived from tobacco chloroplast The vector according to (6) above, wherein the terminator of the identification gene group is a psbA terminator derived from tobacco chloroplasts,
(11) The vector according to any one of (1) to (10) above, wherein the gene for identifying a gene recombinant of the identification gene group is a spectinomycin resistance gene,
(12) The vector according to any one of (1) to (10) above, wherein the gene for identifying a gene recombinant of the identification gene group is an aadA gene having the base sequence represented by SEQ ID NO: 3. ,
(13) Vector pLD6 (FERM P-18260),
(14) A gene recombinant in which a gene encoding a protein is inserted into the vector according to (1) to (13),
(15) A vector comprising a polylinker having a plurality of restriction enzyme sites, an rbcL gene derived from tobacco chloroplasts upstream thereof, and an accD gene derived from tobacco chloroplasts downstream thereof;
(16) The vector according to (15), wherein the polylinker comprises the base sequence represented by SEQ ID NO: 9,
(17) The vector according to (15) or (16), which has a gene comprising a base sequence located at 396-3328 of SEQ ID NO: 8,
(18) Vector pLD200 (FERM P-18261),
(19) The gene recombinant according to (14), wherein (a) a plurality of restriction enzyme sites, a gene encoding a protein, a multicloning region having a ribosome binding site upstream of the gene, A group of constructed genes having a promoter upstream and a terminator downstream of the multicloning region; (b) a gene for identifying a gene recombinant upstream thereof; a promoter having a promoter upstream and a terminator downstream thereof An expression vector, wherein the gene group is inserted into the vector described in (15) to (18) above,
(20) A transformed chloroplast in which the expression vector according to (19) is introduced into a chloroplast,
(21) The transformed chloroplast according to (20), wherein the chloroplast is tobacco chloroplast,
(22) A plant having the transformed chloroplast according to (20) or (21),
(23) The plant according to (22), wherein the plant is tobacco,
(24) a protein obtained from the plant according to (22) or (23),
(25) Gene recombination is prepared by inserting a gene encoding a protein into a region between SphI and EcoRI in the multicloning region of pLD6, and then the gene recombination is cloned. Production of an expression vector characterized by cutting out a gene having a constructed gene and a discriminating gene from the body using NotI and SalI, and inserting the excised gene into a site sandwiched between NotI and SalI of the polylinker of pLD200 Method,
About.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
The vector according to the present invention can be prepared by inserting a pre-construction gene group and a discriminating gene group into a cloning vector.
Here, the cloning vector that can be used in the present invention has (a) a restriction enzyme site into which a pre-construction gene group and a discrimination gene group can be inserted, and (b) can be introduced into a host cell, (C) It is not particularly limited as long as it has the ability to grow in a host cell, and (d) a host cell transformed and introduced with a cloning vector can be specifically detected. Specifically, examples of the cloning vector include plasmids, phages, cosmids, or phagemids. More specifically, plasmids derived from E. coli (eg, pBR322, pBR325, pUC12, etc.), plasmids derived from Bacillus subtilis ( For example, pUB110, pTP5, pC194, etc.), yeast-derived plasmids (eg, pSH19, pSH15, pYES2, etc.), bacteriophages such as λ phage, retroviruses and the like. A commercially available cloning vector may be used.
[0009]
The pre-construction gene group to be inserted into the cloning vector has a multicloning region, a promoter upstream of the multicloning region, and a terminator downstream of the multicloning region.
The multicloning region has a plurality of restriction enzyme sites, an insertion site for inserting a gene encoding a protein sandwiched between two restriction enzyme sites, and a ribosome binding site upstream of the insertion site. Thus, by placing a ribosome binding site upstream of the insertion site for inserting a gene encoding the protein, there is an advantage that the protein can be highly expressed. The ribosome binding site is more preferably about 7 to 11 bases upstream of the translation start point of the gene encoding the protein, and more preferably about 9 bases upstream.
Such a ribosome binding site may have a base sequence known per se that is known to be capable of binding to a ribosome, but an SD sequence is preferred. The SD sequence is an abbreviation for Shine-Dalgarno sequence and is a segment consisting of 4 to 7 nucleotides, and its base sequence is a part or all of 5′-AGGAGGU-3 ′.
The most preferred embodiment of the multicloning region includes a gene having the base sequence set forth in SEQ ID NO: 7.
[0010]
The promoter upstream of the multicloning region may be a known one. Examples include microorganisms (prokaryotes such as Escherichia and Bacillus, eukaryotes such as yeast and filamentous fungi), plant cells, and animal cells. More specifically, examples include Escherichia bacterium trp promoter, trc promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, or T7 promoter; Bacillus genus SPO1 promoter, SPO2 promoter, or penP promoter.
Among them, the promoter is preferably a chloroplast-derived promoter, more preferably a tobacco chloroplast-derived promoter, and most preferably a tobacco chloroplast-derived psbA promoter. The base sequence of the psbA promoter derived from tobacco chloroplast is shown in SEQ ID NO: 5.
[0011]
The terminator downstream of the multicloning region may be known per se. Examples include microorganisms (prokaryotes such as Escherichia and Bacillus, eukaryotes such as yeast and filamentous fungi), plant cells, and animal cells. Among them, the terminator is preferably a chloroplast-derived terminator, more preferably a tobacco chloroplast-derived terminator, and most preferably a tobacco chloroplast-derived rps16 terminator. The base sequence of the rps16 terminator derived from tobacco chloroplast is shown in SEQ ID NO: 6.
[0012]
In the present invention, a discriminating gene group is inserted upstream of the pre-constructed gene group into the cloning vector. The discriminating gene group has a gene for identifying a gene recombinant, a promoter upstream thereof, and a terminator downstream thereof.
The gene for identifying the gene recombinant is not particularly limited, and a gene known per se may be used. Examples include various drug resistance genes or genes that complement the auxotrophy of the host. More specifically, for example, ampicillin resistance gene, neomycin resistance gene (G418 resistance), chloramphenicol resistance gene, kanamycin resistance gene, spectinomycin resistance gene, URA3 gene and the like can be mentioned. Among them, in the present invention, it is preferable to use a spectinomycin resistance gene. The base sequence of the aadA gene, which is the spectinomycin resistance gene, is shown in SEQ ID NO: 3.
[0013]
As the promoter and terminator, those known per se such as those listed as the promoter and terminator used in the pre-constructed gene group may be used. Of these, promoters and terminators derived from chloroplasts are preferred, promoters and terminators derived from tobacco chloroplasts are more preferred, and rrn promoters and psbA terminators derived from tobacco chloroplasts are most preferred. The base sequence of the rrn promoter derived from tobacco chloroplast is shown in SEQ ID NO: 2, and the base sequence of the psbA terminator derived from tobacco chloroplast is shown in SEQ ID NO: 4.
[0014]
The vector may also include one or more nucleic acid sequences that encode one or more factors that favor protein expression, such as activators (eg, trans-acting factors), chaperones, and processing proteases. Further, the vector according to the present invention may have any factor that is functional in the selected host cell.
[0015]
The most preferred embodiment of the vector according to the present invention described above includes pLD6. Such a vector can be easily prepared by the method described in the Examples. In addition, the pLD6 plasmid is based on the Budapest Treaty and is dated March 19, 2001, Institute of Biotechnology, Institute of Industrial Science and Technology, 1-3 1-3 Higashi 1-chome, Tsukuba, Ibaraki, Japan (zip code 305-8666) Is deposited under the deposit number FERM P-18260.
[0016]
A schematic diagram of pLD6 is shown in FIG. The entire base sequence of pLD6 is shown in SEQ ID NO: 1. As can be seen from FIG. 1, pLD6 has (a) a multicloning region having a base sequence represented by SEQ ID NO: 7, and a psbA promoter derived from tobacco chloroplast represented by SEQ ID NO: 5 upstream thereof (SEQ ID NO: 5). 1 and a downstream rps16 terminator derived from tobacco chloroplast represented by SEQ ID NO: 6 (located at 3755-3913 in SEQ ID NO: 1). And (b) an aadA gene (located at 2368-3173 in SEQ ID NO: 1) which is a spectinomycin resistance gene represented by SEQ ID NO: 3 as a gene for identifying a gene recombinant, upstream thereof Upstream of the rrn promoter derived from tobacco chloroplast represented by SEQ ID NO: 2 (located at 2226-2368 in SEQ ID NO: 1), and below And a tobacco chloroplast-derived psbA terminator represented by SEQ ID NO: 4 (located 3175-3568 in SEQ ID NO: 1).
[0017]
A gene encoding a protein is inserted into the multicloning region of the vector according to the present invention to prepare a gene recombinant. For example, in the case of a pLD6 vector, a gene encoding a protein is inserted into a site between SphI and EcoRI in the multicloning region. Hereinafter, the pre-construction gene group into which the gene encoding the protein is inserted in the gene recombinant is referred to as a construction gene group. The protein is a protein desired to be expressed using the expression system according to the present invention, and the type thereof is not particularly limited. Examples thereof include, but are not limited to, a protein having a pharmacological activity, an enzyme necessary for producing a protein useful as a pharmaceutical or industrial material, and the like.
[0018]
Subsequently, the gene recombinant is introduced into an appropriate host cell, the host cell is cultured, and the target gene is cloned. Here, the target genes are the above-described constructed gene group and discriminating gene group (the same applies to the following).
Host cells can be appropriately selected according to the cloning vector, and specific examples include prokaryotes such as Escherichia and Bacillus, eukaryotes such as yeast and filamentous fungi, plant cells, and animal cells. . Moreover, the culture conditions for the host cells may be in accordance with the conditions commonly used in the art depending on the type of host cell. Further, whether or not the target gene has been successfully introduced into the cloned gene can be easily determined based on a selection marker or the like possessed by the cloning vector.
[0019]
In the present invention, the target gene or a gene containing the target gene is then excised from the gene cloned as described above using a restriction enzyme, and inserted into another vector according to the present invention to produce an expression vector. To do. Another vector according to the present invention has a polylinker having a plurality of restriction enzyme sites, an rbcL gene derived from tobacco chloroplasts upstream thereof, and an accD gene derived from tobacco chloroplasts downstream thereof. Is a vector characterized by By doing so, there is an advantage that a gene encoding a foreign protein is easily incorporated into the chromosomal DNA of the host cell by homologous recombination, and the expression level of the protein is increased. Here, the structural gene of the rbcL gene is located at 423-1856 in SEQ ID NO: 8. The structural gene of the accD gene is located at 2624-3328 in SEQ ID NO: 8.
[0020]
As a more preferred embodiment of the vector, a gene comprising a polylinker having a plurality of restriction enzyme sites, an rbcL gene derived from tobacco chloroplasts upstream thereof, and an accD gene derived from tobacco chloroplasts downstream thereof, Examples thereof include vectors that are genes having homology with tobacco chloroplast genes. Here, having homology means having a homology of about 80% or more, preferably about 85% or more, more preferably about 90% or more, and most preferably about 95% or more under highly stringent conditions. Say. The high stringent conditions are, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C., preferably about 60 to 65 ° C. Say. In particular, the most preferable conditions are when the sodium concentration is about 19 mM and the temperature is about 65 ° C.
As a more preferred embodiment of the above vector, a vector in which the polylinker has the base sequence represented by SEQ ID NO: 9 can be mentioned. Among these, a vector having a gene consisting of a base sequence located at SEQ ID NO: 8 at 396-3328 is more preferable.
[0021]
The vector is a cloning vector known per se as described above, a polylinker having a plurality of restriction enzyme sites, preferably a gene having a nucleotide sequence represented by SEQ ID NO: 9, and rbcL derived from tobacco chloroplasts upstream thereof. It is obtained by inserting a gene and an accD gene derived from tobacco chloroplast downstream thereof. Here, since the vector has a plurality of restriction enzyme sites, it has a feature that the target gene or a gene containing it can be easily inserted.
[0022]
The vector may also include one or more nucleic acid sequences that encode one or more factors that favor protein expression, such as activators (eg, trans-acting factors), chaperones, and processing proteases. Further, the vector according to the present invention may have any factor that is functional in the selected host cell.
[0023]
The most preferred embodiment of the vector is pLD200. Such a vector can be easily prepared by the method described in the Examples. In addition, the pLD200 plasmid is based on the Budapest Treaty and is dated March 19, 2001, Institute of Biotechnology, Institute of Industrial Science and Technology, 1-3 1-3 Higashi 1-chome, Tsukuba, Ibaraki, Japan (zip code 305-8666) Is deposited under the deposit number FERM P-18261.
[0024]
A schematic diagram of pLD200 is shown in FIG. As is clear from FIG. 5, pLD200 is a polylinker having a nucleotide sequence represented by (a) SEQ ID NO: 9 in pUC19 (Messing J, Methods in Enzymology, 101: 20 (1983)), which is a known plasmid. (B) an rbcL gene derived from tobacco chloroplasts is inserted upstream thereof, and (c) an accD gene derived from tobacco chloroplasts is inserted downstream thereof.
In addition, the entire base sequence of pLD200 is shown in SEQ ID NO: 8. The polylinker is located at 2125-2145 of SEQ ID NO: 8. The genes located at 396-2124 and 2146-3324 in SEQ ID NO: 8 are tobacco chloroplast-derived genes, and the genes located at 1-395 and 3325-5581 are pUC19-derived genes.
[0025]
As a more preferable method for producing the above expression vector, a gene recombinant is prepared by inserting a gene encoding a protein into a site between SphI and EcoRI of the multicloning region of pLD6, and then the gene recombinant is prepared. After cloning, a gene having a construction gene and a discriminating gene is cut out from the genetic recombinant using NotI and SalI, and the cut out gene is inserted into a site sandwiched between NotI and SalI of the polylinker of pLD200. A method is mentioned.
[0026]
The expression vector thus prepared is introduced into a host cell to produce a transformant. At this time, the host cell is preferably a plant cell, more preferably a chloroplast, and even more preferably a tobacco chloroplast. Thus, by using plant cells as host cells, it is possible to prevent contamination of toxins during the purification process of the protein expressed as described above, and there is an advantage that the cost for protein expression is low. Among them, by using chloroplasts as host cells, the protein encoded by the introduced gene can be highly expressed as described above, and further, scattering to the environment via pollen of the introduced gene can be prevented. There are advantages such as.
[0027]
As a method for transforming by introducing the expression vector into a host cell, a known method may be used. For example, a particle gun method may be used in which the expression vector is applied to very fine particles of gold or tungsten, the particles to which the expression vector is attached are injected into a host cell with an explosive or high-pressure gas, and the expression vector is introduced. Among them, the gene transfer system to higher plant chloroplasts is based on the particle gun technique (Svab, Z., Hajdukiewicz, P., and Maliga, P., Stable transformation of plastids in higher plants.Proc. Natl. Acad. Sci. USA,87, 8526-8530 (1990)) or PEG technique (Golds, T., Maliga, P., and Koop, H.-U., Stable plastid transformation in PEG-treated protoplasts ofNicotiana tabacum. Bio / Technol.,1195-97 (1993)).
[0028]
The plant having the above-mentioned transformed chloroplast according to the present invention can be obtained by a method known per se. Here, although the said plant is not specifically limited, A higher plant is preferable and a tobacco is more preferable.
Next, the plant is grown under known conditions according to the plant, and the target protein is purified from the plant body by a method known per se. Protein purification can be performed by appropriately combining per se known separation and purification methods. As these known separation and purification methods, there are mainly differences in molecular weight such as methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration or SDS-polyacrylamide gel electrophoresis. A method utilizing charge difference such as ion exchange chromatography; a method utilizing specific affinity such as affinity chromatography; a method utilizing hydrophobic difference such as reverse phase high performance liquid chromatography; A method using the difference in isoelectric point, such as isoelectric focusing, is used.
In this way, it is possible to produce a large amount of useful proteins such as proteins having pharmacological activity and industrial enzymes, and contamination of toxins is substantially recognized during the purification of expressed proteins as in the expression system using E. coli. There is an advantage that it is not possible. Furthermore, there is an advantage that the target protein can be produced at a lower cost and lower energy than the expression system using animal cells.
[0029]
The basic operation of genetic engineering or bioengineering described above is a commercially available experiment, for example, Molecular Cloning published in 1982, Cold Spring Harbor Laboratory, published in 1989. Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory, and the like.
[0030]
【Example】
[Step 1; Production of pLD6]
A multicloning region consisting of the base sequence represented by SEQ ID NO: 7 is placed downstream of the psbA promoter derived from tobacco chloroplasts, and a terminator (Trps16) of the ribosomal protein rps16 derived from tobacco chloroplasts is placed downstream of the psbA promoter The constructed plasmid was constructed. In this multicloning region, the ribosome binding site (SD sequence; 5 nucleotides starting from the 13th position counted from the 5 ′ end of the base sequence shown in FIG. A segment consisting of: An identification gene group (aadA cassette) for selection of chloroplast transformants was placed upstream of these pre-construction gene groups. Both of these gene groups were cut out with NotI-SalI. The vector thus constructed was named pLD6 (FIG. 1).
A more detailed construction process is shown in FIGS. pBluescript II SK (+) (Gene Bank Accession Number: X52328), the starting material for the construction of pLD6, was purchased from Stratagene. Further, the synthetic DNA of FIG. 8 was chemically synthesized using a DNA synthesizer model 392 (manufactured by Perkin Elmer Co., Ltd.).
[0031]
[Step 2: Production of gene recombinants]
GFP is used as a reporter gene (Sidorov, VA, Kasten, D., Pang, S.-Z., Hajdukiewicz, PTJ, Staub, JM, and Nehra, NS, Stable chloroplast transformation in potato: use of green fluorescent protein as a plastid marker.Plant J,19209-216 (1999)), the gene encoding the GFP was inserted into the multi-cloning region of pLD6 (the site whose nucleotide sequence is described in FIG. 1) between SphI and EcoRI.
The gene recombinant was introduced into Escherichia coli, and the Escherichia coli was cultured in LB medium supplemented with spectinomycin at 37 ° C. for 16 hours to select Escherichia coli into which the gene recombinant was introduced. The selected Escherichia coli was cultured in LB medium at 37 ° C. for 16 hours, centrifuged after culturing, the cells were collected, and the gene recombinant (plasmid DNA) was purified according to a conventional method. The composition in 1 L of LB medium is 10 g tryptone, 5 g yeast extract, and 5 g NaCl.
[0032]
[Step 3; Production of pLD200]
In addition, a vector pLD200 was constructed in which a homologous sequence of the tobacco chloroplast gene containing the rbcL gene and the accD gene (base sequence located at 396-3328 of SEQ ID NO: 8) was introduced upstream of NotI and downstream of SalI. (FIG. 5). A more detailed construction process is shown in FIG. pUC19 (Gene Bank Accession Number: L09736), the starting material for the construction of pLD200, was sold by the author of Messing J, Methods in Enzymology, 101: 20 (1983). The synthetic DNA of FIG. 6 was chemically synthesized using a DNA synthesizer model 392 (manufactured by Perkin Elmer Co., Ltd.).
[0033]
[Step 4: Preparation of expression vector]
The gene recombinant (purified DNA) obtained in step 2 was digested with NotI and SalI. PLD200 is also digested with NotI and SalI and sandwiched between NotI and SalI of a polylinker (a segment consisting of 21 nucleotides starting from the 5th position counting from the 5 ′ end of the site where the nucleotide sequence is described in FIG. 6). The digested product of NotI and SalI of the recombinant gene (purified DNA) was inserted into the obtained portion. In this way, an expression vector according to the present invention was prepared.
[0034]
[Step 5: Production of transformed chloroplasts]
The obtained expression vector was introduced into tobacco chloroplasts to produce transformed chloroplasts. Tobacco chloroplast transformation is a known method (Svab, Z., Hajdukiewicz, P., and Maliga, P., Stable transformation of plastids in higher plants.Proc. Natl. Acad. Sci. USA, 87, 8526-8530 (1990)).
[0035]
The appearance of the chloroplast transformant was the same as that of the wild species (FIG. 2), but as a result of observation under a fluorescence microscope, strong GFP-derived fluorescence not seen in the wild species was observed (FIG. 3). . Further, when protein was extracted from this transformant and subjected to SDS-PAGE, a band having a high molecular weight corresponding to GFP was confirmed (FIG. 4). From the densitometry of the density of this band, the total expression amount was about 20% of the total soluble fraction.
[0036]
【The invention's effect】
Unlike the heterologous protein expression system using E. coli, the heterologous protein expression system using the plant according to the present invention has the advantage that no toxin is mixed in during purification of the expressed protein. . In addition, since plants grow by photosynthesis, energy input for plant growth can be reduced, and as a result, there is an advantage that a product (protein) can be produced at low cost.
[0037]
As a concern of conventional gene recombination technology, there is environmental pollution by artificially manipulated genes. This is a concern that the introduced artificially modified gene spreads to the environment by crossing, mating, and the like. For this reason, biological containment during genetic recombination has been emphasized. In the present invention, as a preferred embodiment, a gene is introduced into a chloroplast to express a protein. Since gene introduction into such chloroplasts is maternally inherited from the chloroplast gene, the present invention is useful from the viewpoint of environmental conservation in terms of biological containment in natural phenomena.
[0038]
In the present invention, as a preferred embodiment, a gene is introduced into a chloroplast as described above to express a protein. Since such a chloroplast gene expression system is a prokaryotic cell type, it can be easily transferred from an E. coli prokaryotic microorganism. As a result, there is an advantage that the migration from the mass expression system of heterologous proteins currently performed in Escherichia coli and the like can be performed promptly.
[0039]
The protein expression system according to the present invention includes, for example, production of pharmaceuticals, industrial enzymes, polyester, biodegradable plastics, oils, fats, proteins, hydrocarbons or terpenes, livestock feed (proteins, lipids, carbohydrates) It is useful for preparing a plant body that can be easily digested and absorbed by directly adjusting the ingredients, and improving the variety of foliage plants and creating a complex environmental stress resistant plant.
[0040]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows a schematic diagram of pLD6. Prrn represents the rrn promoter derived from tobacco chloroplasts. The rrn promoter has a base sequence represented by SEQ ID NO: 2 and is located at 2226-2368 in SEQ ID NO: 1. aadA represents a spectinomycin resistance gene. The spectinomycin resistance gene has a base sequence represented by SEQ ID NO: 3 and is located at 2368-3173 in SEQ ID NO: 1. TpsbA represents a psbA terminator derived from tobacco chloroplasts. The psbA terminator has a base sequence represented by SEQ ID NO: 4 and is located at 3175-3568 in SEQ ID NO: 1. PpsbA represents the psbA promoter derived from tobacco chloroplasts. The psbA promoter has a base sequence represented by SEQ ID NO: 5 and is located at 3569-3701 in SEQ ID NO: 1. Trps16 represents an rps16 terminator derived from tobacco chloroplasts. The rps16 terminator has a base sequence represented by SEQ ID NO: 6 and is located at 3755-3913 in SEQ ID NO: 1. The site describing the base sequence is a multicloning region. The remaining part (thick line part) is a gene derived from pBluescript II SK (+). BglII, SphI, ClaI and EcoRI represent restriction enzyme sites, and SD represents an SD sequence (a segment consisting of 5 nucleotides starting from the 13th position counting from the 5 ′ end of the base sequence shown in FIG. 1).
FIG. 2 shows the appearance of tobacco having a chloroplast transformant obtained in an example in which the GFP gene is expressed.
FIG. 3 shows a fluorescence image of a tobacco leaf having a chloroplast transformant obtained in an example in which the GFP gene is expressed.
FIG. 4 was obtained by extracting proteins from tobacco having chloroplast transformants obtained in Example and performing SDS-PAGE (Panel A). Similarly, SDS-PAGE was performed using wild-type tobacco into which no GFP gene was introduced (Panel B). Accumulation of GFP in an amount comparable to RuBisCO (rbcL + rbcS), which accounts for 50% of the total soluble protein amount, was observed in the GFP transgenic tobacco obtained in the examples. Panel M is a molecular weight marker.
FIG. 5 shows a schematic diagram of pLD200. rbcL represents the rbcL gene derived from tobacco chloroplasts. accD represents the accD gene derived from tobacco chloroplasts. Among the sites describing the base sequence, the 5th to 21st bases counted from the 5 ′ end are polylinkers. The bold line portion is a gene derived from pUC19. NotI, NheI and SphI represent restriction enzyme sites.
FIG. 6 shows the construction process of pLD200.
FIG. 7 shows the first step in the construction process of pLD6.
FIG. 8 shows the second step of the construction process of pLD6.
FIG. 9 shows a third step in the construction process of pLD6.

Claims (15)

From upstream,
(A) an rbcL gene derived from tobacco chloroplasts,
(B) a gene for identifying a gene recombinant, a group of identification genes having a promoter upstream and a terminator downstream thereof,
(C) a psbA promoter derived from tobacco chloroplasts,
(D) a plurality of restriction enzyme sites, an insertion site into which a gene encoding a protein sandwiched between two restriction enzyme sites can be inserted, and a multicloning region having a ribosome binding site upstream of the insertion site,
( E ) a vector having an rps16 terminator derived from tobacco chloroplasts, and ( f ) an accD gene derived from tobacco chloroplasts.   The vector according to claim 1, wherein the multicloning region has a ribosome binding site 7 to 11 bases upstream of the translation start point of the gene encoding the protein.   The vector according to claim 1 or 2, wherein the ribosome binding site is an SD sequence.   The vector according to any one of claims 1 to 3, wherein the multicloning region consists of a base sequence represented by SEQ ID NO: 7.   The vector according to any one of claims 1 to 4, wherein the promoter of the discriminating gene group is an rrn promoter derived from tobacco chloroplast, and the terminator of the discriminating gene group is a psbA terminator derived from tobacco chloroplast. (A) The vector according to any one of claims 1 to 5, wherein the rbcL gene derived from tobacco chloroplast is a gene consisting of the nucleotide sequence of nucleotide numbers 423 to 1856 of SEQ ID NO: 8. (C) The vector according to any one of claims 1 to 6, wherein the psbA promoter derived from tobacco chloroplast is a gene consisting of the nucleotide sequence of SEQ ID NO: 5. (D) The vector according to any one of claims 1 to 7, wherein the rps16 terminator derived from tobacco chloroplast is a gene consisting of the nucleotide sequence of SEQ ID NO: 6. (E) The vector according to any one of claims 1 to 8, wherein the accD gene derived from tobacco chloroplast is a gene consisting of a base sequence represented by base numbers 2624 to 3328 of SEQ ID NO: 8. A gene recombinant in which a gene encoding a protein is inserted into the vector according to any one of claims 1 to 9. A transformed chloroplast in which the genetic recombinant according to claim 10 is introduced into a chloroplast. The transformed chloroplast according to claim 11, wherein the chloroplast is tobacco chloroplast. A plant having the transformed chloroplast according to claim 11 or 12. The plant according to claim 13, wherein the plant is tobacco. A method for producing a protein , wherein the plant according to claim 13 or 14 is grown and the protein encoded by the gene inserted into the vector according to any one of claims 1 to 9 is separated .

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