JP2004113205A - Temperature sensitive replication factor and plasmid containing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a new plasmid overcoming both lower level continuous gene expression which a high copy number plasmid vector has and the instability of an expressed vector caused by using a vector having a lower copy number to avoid the above gene expression and capable of using a conventional IPTG addition method inspite of virus infection in beginning of expression by switching on a promoter, and provide a gene expression method, and the like using the new plasmid. <P>SOLUTION: This invention relates to a pSC101DNA replication factor (RepA) mutant in which the 13th amino acid in the amino acid sequence of the RepA mutates to threonine and the 56th amino acid to valine and having function of temperature sensitive replication and temperature sensitive separation, a gene for coding the same, a plasmid containing the gene, a cell transformed with the plasmid and a method for producing a protein coded with a foreign gene using the cells. <P>COPYRIGHT: (C)2004,JPO

Description

【００２９】
表１中の「ＡＡ３」はＮ末端から３番目のアミノ酸、「ＡＡ１３」はＮ末端から１３番目のアミノ酸、「ＡＡ５６」はＮ末端から５６番目のアミノ酸を示す。「ＤＮＡ複製効率」ｋは、式
ｋ＝２・ｅｘｐ［ｌｎ（ｂ_ｎ／ｃ_ｎ）／（Ｎ−ｎ）］−１
で与えられるものであり（各パラメーターについてはＨａｓｈｉｍｏｔｏ−Ｇｏｔｏｈ　ｅｔ　ａｌ．，　Ｇｅｎｅ，　２４１，Ｐ１８５−１９１を参照）、表中の数値は高温（４２℃）での低温（３０℃）に対する相対的ＤＮＡ複製効率として表したものである。図１におけるプラスミドを含む宿主菌の増殖速度について、高温側の直線の傾きを低温側の直線の傾きで割った値に相当する。「コロニー形成能」は、プラスミド保持菌が４２℃で形成するコロニー数を、３０℃で形成するコロニー数で割った値である。「コピー数」は、低温（３０℃）での宿主細胞の染色体数当たりのプラスミドのコピー数である。下線で示したプラスミドはｒｅｐＡ遺伝子のｍＲＮＡの半減期を調節すると推量される３’−非翻訳領域を欠失している。括弧で示されたｐＴＨ１９ｋｓｓｈは３０℃でもカナマイシン耐性菌の増殖はほとんど見られなかった。
【００３０】
【発明の効果】
本発明のプラスミドベクターは、鋭敏な温度感受性を有し、培養温度の変更操作により、（１）宿主菌が４２℃において増殖中にプラスミド複製が起らないので選択薬剤下に宿主菌細胞が増殖せず、カナマイシン耐性コロニーを形成した大腸菌宿主細胞がプラスミドを染色体に組み込んだものしか存在しえないこと、（２）その様な大腸菌ではそのプラスミドに乗っていたラクトースプロモーターは染色体遺伝子のｌａｃＩ^ｑによって完全に抑制されること、（３）また、一旦組み込まれたプラスミドは３７℃以上の培養条件では染色体よりきりだされることなく染色体ＤＮＡの一部として安定に子孫細胞に伝達されること、（４）十分に増殖した細胞培養液にＩＰＴＧを加えることによりラクトースプロモーターのスィッチを入れることにより、宿主細胞に毒性を示す蛋白質の工業的生産を効率的に行うことができる。宿主に用いる大腸菌としてはｒｅｃＡ^＋、ｏｍｐＴ⁻、ｌａｃＩ^ｑ、ΔｌａｃＵ１６９の遺伝子型を持っていることが望ましい。それぞれの遺伝子は、プラスミドの染色体への組込み、組換え蛋白質の分解酵素、ラクトースプロモーターのスイッチオフ、ＩＰＴＧ添加時に組換え蛋白質遺伝子のラクトースプロモーター以外に、野生型大腸菌が持っているｄｅ　ｎｏｖｏのｌａｃプロモーターがスイッチオンされることの防止のために有用である。これらの工程により、細胞にとって細胞毒性の強い物質を発現させることが可能となり、大腸菌のような原核細胞による遺伝子組換えタンパク質の工業的な生産を可能とするものである。
【００３１】
【配列表】

【図面の簡単な説明】
【図１】図１は、複製についての温度感受性プラスミドｐＴＨ１９ｋｓ１ｈ１４（図１Ａ）、分離についての温度感受性プラスミドｐＴＨ１９ｋｓ５（図１Ｂ）、及び本発明のプラスミドｐＴＨ１４ｋｓｓｈ（図１Ｃ）を含有する細胞の、低温（３０℃）（図１中の丸印及び黒丸印を付した線）及び高温（４１．５℃）（図１中の四角印及び黒四角印を付した線）での増殖速度をグラフ化したものである。図１のそれぞれの縦軸は菌数／ｍｌの対数、横軸は培養時間（時間）を示す。○、３０℃での総細胞数；●、３０℃でのカナマイシン耐性細胞数；□、４２℃での総細胞数；■、４２℃でのカナマイシン耐性細胞数
【図２】図２は、本発明のプラスミドベクターの例を示す。ｓ１、ｓ５及びｈ１４は本発明の変異部位を示し、ｐＢＲ３２２挿入箇所の例が示されている。→、←はｐＢＲ３２２のＤＮＡ複製の方向であり、ｐＴＨ１４ｋｓｓｈと同方向の場合を＋、逆向きの場合を−としている。
【図３】図３は、図２に例示した本発明のプラスミドベクターにおけるｐＳＣ１０１ＤＮＡ複製開始因子変異体（ｒｅｐＡｓｓｈ）の塩基配列と、野生型（ｒｅｐＡｗｔ）の塩基配列の比較を示したものである。塩基の７番目、３７番目及び１６７番目に変異があることを示す。
【図４】図４は、図３の続きの塩基配列の比較を示す。
【図５】図５は、図２に例示した本発明のプラスミドベクターにおけるｐＳＣ１０１ＤＮＡ複製開始因子変異体蛋白質（ＲｅｐＡｓｓｈ）のアミノ酸配列と、野生型蛋白質（ＲｅｐＡｗｔ）のアミノ酸配列の比較を示したものである。アミノ酸の３番目、１３番目及び５６番目に置換があることを示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention provides a pSC101 DNA replication initiation factor (RepA) mutant having a temperature-sensitive replication ability and a temperature-sensitive separation ability, a gene encoding the same, a plasmid containing the gene, a cell transformed with the plasmid, The present invention relates to a method for producing a protein encoded by a foreign gene using the cells.
Since the pSC101 DNA replication initiation factor (RepA) mutant of the present invention has a temperature-sensitive replication ability and a temperature-sensitive separation ability, replication and separation (distribution) of a plasmid having the gene of the mutant are required for expression of a foreign gene. Must be performed at a temperature different from the culture temperature. Therefore, the steps of replication and isolation of the vector in the host cell and the step of expressing the foreign gene in the host cell are performed as different steps.
The present invention provides a method for efficiently producing a protein using a recombinant gene. More specifically, the present invention provides a novel industrial method for producing a cytotoxic protein.
[0002]
[Prior art]
When producing industrially useful proteins or proteinaceous pharmaceuticals using recombinant genes, host cells can be broadly divided into prokaryotic cells such as Escherichia coli, yeast cells, animal cells, and plant cells. There is a method using eukaryotic cells such as cells. The choice of one of these is based on the various properties of the particular protein. In general, a method using Escherichia coli is desirable because it has high industrial cost performance and can be produced and purified at low cost. However, industrially or medically useful proteins are often derived from genes of higher organisms such as humans, and are highly heterogeneous in cells of organisms that are taxonomically too distant, such as Escherichia coli. In the high expression system Escherichia coli host vector system, the cytotoxicity of the protein often makes it impossible to mass-cultivate Escherichia coli at an industrial level. In general, plasmid vectors of Escherichia coli used industrially have a large number of copies in a cell, and suppression in a gene expression control device (promoter operator, hereinafter abbreviated as PO) used does not completely function. Often, unintended expression is seen at the basal level. In addition, a protein having cytotoxicity that cannot be ignored in host cells even at such a low level of expression makes industrial production in E. coli impossible. In such a case, the expression should be switched on and off so that expression of the foreign gene in the growing Escherichia coli is minimized and expression of the foreign gene is performed only when the cell concentration is sufficiently increased. It needs to be controlled functionally. For this purpose, plasmid vectors for various expression systems have been developed. However, if the cytotoxicity is so strong that it cannot be ignored, production in Escherichia coli may not be possible.
[0003]
As described above, one of the factors that makes it difficult to avoid cytotoxicity due to the protein to be expressed is that it is difficult to completely switch off the function of the expression control region (PO region) of the gene on the expression vector. is there. In order to express a gene for the purpose of obtaining a protein, it is necessary to turn on the promoter after growing Escherichia coli to a predetermined concentration. This is because, when a foreign gene is expressed in a state where the concentration of E. coli is low, the growth of E. coli is inhibited, and the protein production efficiency is reduced. As such a control means, for example, there is a method using a lactose promoter-operator (POlac) and its repressor gene (lacI). This method uses the lacI gene as its repressor gene. ^q The promoter is switched off by overexpression by mutation, and IPTG, a repressor inactivating factor, is dissolved in the culture solution and switched on. However, when the vector carrying the gene to be expressed downstream of POlac is a so-called high copy number vector having a high copy number per cell, it is necessary to completely switch off even the overexpressed repressor. Is difficult and expression at low levels usually occurs continuously. In addition, the lac promoter commonly used in expression vectors is a mutant called lacUV5, and it is known that low-level expression proceeds even in the presence of a repressor. Under these circumstances, a protein that exhibits strong cytotoxicity even at low levels of expression causes growth inhibition of Escherichia coli, which is fatal for industrial production of recombinant proteins. In an extreme case, it was impossible to construct an expression vector as well as to produce a protein.
[0004]
As an improved method for producing such a toxic protein, a method using a pET plasmid vector system is commercially available from Novagen (Novagen, Inc., 601 Science Drive, Madison, W153711, USA). This system is characterized by using a T7 phage promoter as a promoter of a gene to be expressed. Since normal Escherichia coli does not produce any T7 RNA polymerase, even if a highly toxic gene is placed on this vector, the gene is not expressed at all and the Escherichia coli is not inhibited from growing. Therefore, construction of an expression vector for the gene can be performed without any problem. However, in order to express the gene, it must be transferred to a special E. coli strain BL21 (DE3) that expresses the T7 RNA polymerase gene. This E. coli strain (F ⁻ , OmpT, hsdSB (rB ⁻ mB ⁻ ), Gal (lcI857, ind1, Sam7, nin5, lacUV5-T7 gene1), dcm (DE3)), the T7 RNA polymerase gene is inserted into the chromosomal DNA of E. coli under the control of the lacUV5 promoter. Even during growth prior to IPTG administration, production of low levels of T7 RNA polymerase under the control of the lacUV5 promoter is unavoidable, and as a result, low levels of recombinant protein are continuously produced from genes on high copy number plasmids, If the toxicity of is high, growth inhibition is inevitable. To avoid this, Novagen also offers a method for expression using normal E. coli that does not produce T7 RNA polymerase, but this method uses a large amount of virus to supply T7 RNA polymerase by viral infection. And the cell concentration of E. coli must be reduced to several hundredths or more, which is not suitable for practical and industrial production of proteins.
[0005]
On the other hand, when a low copy number vector is used, spontaneous dropout of the vector accompanying cell division usually occurs frequently even in selective culture containing an antibiotic, resulting in extremely low gene expression efficiency.
As described above, when producing a toxic protein in Escherichia coli, it is a problem that high-copy-number plasmid vectors cannot completely suppress gene expression during cell growth in the previous stage. If a low-copy-number plasmid vector is used, vector instability becomes a problem.
The present inventors have developed a plasmid having a temperature-sensitive mutation to solve such a problem (T. Hashimoto, et al., J. Bacteriol., 127, 1561-1563 (1976); T. Hashimoto-Gotoh, et al., J. Bacteriol., 131 (2), 405-412 (1977)). These temperature-sensitive plasmids were obtained by mutating the tetracycline-resistant R plasmid pSC101 with hydroxylamine, and were classified into the following three types. Type I plasmids (pHSG1, pHSG2, pHSG4, pHSG6, pHSG7, and pHSG17; hereinafter, the plasmids described as "pHS" in the above two reports were later renamed "pHSG. J. Bacteriol., 1979." 139, pp. 597-607), once the host cells were cultured at 42 ° C. in the absence of tetracycline, the colony was formed in the presence of tetracycline even when the temperature was returned to 30 ° C. or kept at 42 ° C. He lost his ability. With the type II plasmid (pHSG3), regardless of the pre-incubation temperature, the host bacteria did not form colonies at 42 ° C in a medium containing tetracycline, but had a colony-forming ability at 30 ° C. When the type III plasmids (pHSG5, pHSG8, and pHSG16) were preincubated at 42 ° C in the absence of tetracycline, the host cells could not grow on a tetracycline-containing medium even after returning to 30 ° C. When incubated, it had a colony forming ability even at 42 ° C.
Among these, plasmid pHSG1 completely inhibited DNA replication, particularly at 42 ° C. For example, replication was completely stable at 30 ° C. and transmitted to 100% of offspring, but could not be replicated at high temperatures (42 ° C.).
This temperature-sensitive replication (ts-Rep) plasmid is a plasmid in which plasmid replication is only partially inhibited at the optimal temperature of cell culture for protein production (around 37 ° C.) and shows incomplete temperature sensitivity. Met.
[0006]
On the other hand, in the case of the temperature-sensitive plasmid (ts-Seg), a phenomenon called segregation occurs, and DNA replication can be performed at a high temperature. However, the distribution of the plasmid copy to the two daughter cells is equally distributed to the grown cells. I knew it would not happen. Isolation is a phenomenon in which a plasmid is not distributed to a portion of its daughter cells after the host cell divides, but is progressively lost from progeny cells. In the plasmid, the daughter plasmid copy produced by replication of the plasmid was frequently lost from progeny cells produced by the division (proliferation) of the host cell under high-temperature (40 to 42 ° C.) culture of the host cell. . When such a separation occurs, cells containing the foreign gene cloned in the plasmid are gradually diluted with respect to the whole cultured cells, and the productivity of the protein per culture solution is significantly reduced. For example, pHSG5, a kind of the above-mentioned temperature-sensitive isolation plasmid (ts-Seg), can replicate at 43 ° C., but 35% of daughter cells carry the plasmid every time the cell containing the plasmid divides. It did not. In addition, pHSG5 had a low copy number even at low temperature (30 ° C.), and the result was that pSC101 and pHSG1 had 5 copy numbers per chromosome of the host cell, while pHSG5 had only 1 copy number.
Thus, the present inventors have succeeded in developing expression vectors for a temperature-sensitive replication plasmid and a temperature-sensitive separation plasmid (T. Hashimoto-Gotoh, et al., Gene, 241, 185-191 (2000)). As a result, plasmids (ts-Rep) derived from pHSG1 and having temperature sensitivity for replication (ts-Rep) pTH18cs1, pTH19cs1, pTH18ks1 and pTH19ks1 were developed, and plasmids derived from pHSG5 and having temperature sensitivity for separation (ts-Seg) pTH18cs5, pTH19cs5, pTH18ks5, and pTH19ks5 were developed. The cs1 (chloramphenicol-resistant) and ks1 (kanamycin-resistant) plasmids whose replication is temperature-sensitive have an extra nucleotide sequence (ACGT) that is cleaved by the restriction enzyme MaeII in the coding region of the repA gene. (There are two places where the wild type has only one place in the entire coding region). Similarly, in the case of the cs5- and ks5-type plasmids in which the separation is temperature-sensitive, the restriction enzyme MaeI which should be in the coding region near the amino terminus is used. It was found that the base sequence to be cleaved (CTAG) was lost (only three wild-type sites were possessed in the entire coding region).
These plasmids are temperature-sensitive for replication or isolation, respectively, and can only control either replication or isolation by controlling temperature. Moreover, the relative colony-forming ability in the presence of antibiotics was 10 ° C at 42 ° C. ^-4 Almost 100% of the cells formed colonies at a temperature (around 37 ° C.) under the culture conditions for producing the protein, although the degree of reduction was to a certain extent. On the other hand, almost 100% of the cells of the temperature-sensitive isolation plasmid retained colony forming ability under the same conditions even at 42 ° C. Therefore, these expression vectors were not always practical. From an industrial point of view, it has been desired to develop a plasmid vector in which replication and separation as a plasmid are simultaneously suppressed even at around 37 ° C., and are replicated as part of a chromosome-integrated state.
[0007]
[Non-patent document 1]
T. Hashimoto, et al. J. et al. Bacteriol. , 127, 1561-1563 (1976);
[Non-patent document 2]
T. Hashimoto-Gotoh, et al. J. et al. Bacteriol. , 131 (2), 405-412 (1977).
[Non-Patent Document 3]
Hamilton et al. J. et al. Bacteriol. 171: 4617-4622, (1989).
[Non-patent document 4]
T. Hashimoto-Gotoh, et al. , Gene, 241, 185-191 (2000).
[0008]
[Problems to be solved by the invention]
As described above, in the industrial production of proteins by genetic recombination technology using Escherichia coli, conventionally, (1) "gene amplification" which replicates expression vectors by culturing host cells and stably distributes them to progeny cells Step and the “gene expression step” for expressing a useful substance occur simultaneously, and in some cases, the host and product of the expression system are degraded. It was a major obstacle. This is particularly acute when the protein exhibits some toxicity to host bacterial cells, and in many cases, production of recombinant proteins by Escherichia coli has been substantially impossible. For this reason, there has been a demand for the development of a host vector system capable of improving the production efficiency of a protein having toxicity to Escherichia coli.
The present invention provides a novel plasmid capable of controlling the replication and separation of the plasmid by temperature, and provides a novel expression vector capable of mass-producing a useful substance by genetic recombination technology.
Furthermore, it is an object of the present invention to: 1) low-level, sustained gene expression of high-copy-number plasmid vectors; 2) instability of expression vectors caused by using low-copy-number plasmid vectors to avoid them. It is an object of the present invention to provide a method that can overcome both the problems of the above-mentioned methods and can use the conventional IPTG addition method instead of virus infection to initiate expression when the promoter is switched on.
[0009]
[Means for Solving the Problems]
In view of the above situation, the present inventor inserts a plasmid vector that expresses the gene of a recombinant protein under the control of a lactose promoter-operator (POlac) into the chromosome of the host E. coli under specific conditions and stably maintains it. A pSC101-based plasmid vector having a temperature-sensitive double mutation in the DNA synthesis initiation protein gene (repA) was successfully constructed.
That is, the present invention relates to the pSC101 DNA replication initiation factor (RepA), in which alanine, the 13th amino acid in the amino acid sequence, is mutated to threonine, and alanine, the 56th amino acid, is mutated to valine. PSC101 DNA replication initiation factor mutant (RepAs1s5 or simply RepAss) having characteristic temperature-sensitive replication ability and temperature-sensitive separation ability, preferably glutamic acid, which is the third amino acid in the amino acid sequence, is also mutated to lysine. PSC101 DNA replication initiation factor mutant (RepAs1s5h14 or simply RepAssh) having a temperature-sensitive replication ability and a temperature-sensitive separation ability. More preferably, the pSC101 DNA replication initiation factor gene (repA) has a pSC101 DNA replication initiation factor mutant having the amino acid sequence shown in SEQ ID NO: 1 or 2 in the sequence listing ( Rep Ass, or Rep ASH).
The present invention also relates to a gene (repA) encoding pSC101 DNA replication initiation factor (RepA), wherein guanine (G), the 37th base of the translation region, is mutated to adenine (A), and A gene encoding a pSC101 DNA replication initiation factor mutant (RepAss) having a temperature-sensitive replication ability and a temperature-sensitive separation ability, characterized in that cytosine (C), which is a base of, is mutated to thymine (T), preferably A pSC101 DNA replication initiation factor mutant having a temperature-sensitive replication ability and a temperature-sensitive separability, wherein guanine (G), the seventh base of the translation region, is mutated to adenine (A). RepAssh). More preferably, the pSC101 DNA replication initiation factor mutant (RepA
) Relates to a gene (repAss or repAssh) encoding a pSC101 DNA replication initiation factor mutant (RepAss or RepAssh) having the nucleotide sequence shown in SEQ ID NO: 3 or 4 in the sequence listing.
[0010]
Further, the present invention provides a plasmid vector containing a gene (repAss or repAssh) encoding the above-described pSC101 DNA replication initiation factor mutant (RepAss or RepAssh), preferably a plasmid vector under the control of a lactose promoter-operator (POlac). A plasmid vector that expresses the gene for the recombinant protein, more preferably a plasmid vector derived from pSC101, and even more preferably a base that regulates the half-life of mRNA downstream of the gene encoding the pSC101 DNA replication initiator mutant (RepAss or RepAssh). It relates to a plasmid vector having a sequence.
Furthermore, the present invention relates to a cell transformed with the above-mentioned plasmid vector, preferably Escherichia coli.
The present invention provides a method for cell replication, wherein the replication and isolation of the above-described plasmid are performed at a temperature different from the cell culture temperature, and the plasmid replication and isolation are not substantially performed at the cell culture temperature. The present invention relates to a culture method, preferably a culture method in which the temperature for replication and isolation as a plasmid is 28 to 33 ° C, and the replication in the state of being inserted into the E. coli chromosome is 35 to 42 ° C.
The present invention also provides a cell transformed with the above-described plasmid vector into which a foreign gene has been inserted, by replicating and separating the above-described plasmid at a temperature different from the cell culturing temperature, -42 ° C.), a method for producing a protein encoded by the foreign gene by a cell culture method characterized in that the vector is not replicated and separated as a plasmid, preferably a temperature for replication and separation of the plasmid. Is 28 to 33 ° C, and a culture temperature for expression of the foreign gene is 35 to 42 ° C.
[0011]
The present inventors have developed temperature-sensitive (ts-Rep) plasmids pTH18cs1, pTH19cs1, pTH18ks1, and pTH19ks1 for replication and have temperature-sensitive (ts-Seg) plasmids pTH18cs5, pTH19cs5, pTH18ks5, and (Ts-Seg) for isolation. pTH19ks5 has been developed (T. Hashimoto-Gotoh, et al., Gene, 241, 185-191 (2000)).
These plasmids are all derived from the pSC101 plasmid with respect to DNA replication, and are replication-sensitive plasmids of the cs1 type (chloramphenicol resistance) and the ks1 type (kanamycin resistance), and separation at temperature These are sensitive cs5 (chloramphenicol resistant) and ks5 (kanamycin resistant) plasmids.
A plasmid having a temperature sensitivity for replication (temperature-sensitive replication plasmid (ts-Rep vector) = temperature-sensitive replication-vectors: ts-Rep) has a replication ability that is temperature-sensitive and can replicate at a low temperature, for example, 30 ° C. However, replication cannot be performed at a high temperature, for example, at 41.5 ° C. In addition, the temperature-sensitive plasmid for separation (temperature-sensitive separation plasmid (ts-Seg vector) = temperature sensitive-segmentation plasmid vector: ts-Seg) has a temperature-sensitive separation ability at 30 ° C. Although stable transmission to daughter cells is possible, unstable separation occurs at 41.5 ° C.
However, these plasmids were capable of controlling the temperature for replication or separation.However, for mutants in which both were introduced into the same gene at the same time, whether or not they can coexist, and what phenotype if possible Had not been considered.
[0012]
For example, the growth rate of Escherichia coli in the absence of selective antibiotics at each culture temperature for plasmids that are temperature sensitive for replication (ts-Rep) and for plasmids that are temperature sensitive for isolation (ts-Seg) is When viewed, they are as shown in FIGS. 1A and 1B, respectively. In the graph of FIG. 1, the vertical axis indicates the number of E. coli per unit volume by logarithm, and the horizontal axis indicates the culture time. The line with a circle (印 or ●) indicates the case of culturing at a low temperature (eg, 30 ° C.), and the line with a square (□ or Δ) indicates the culture at a high temperature (eg, 41.5 ° C.). This shows the case of culture. The total number of cells is shown on the vertical axis per unit volume, and the actual measurement is performed by appropriately diluting an agar medium containing no selective antibiotics. On the other hand, black marks (● or Δ) indicate the case where the measurement was performed using an agar medium containing an antibiotic. This indicates the number of cells containing the plasmid.
In the case of the ts-Rep plasmid, whose replication is temperature-sensitive (FIG. 1A), the number of bacteria increases semi-logarithmically with time in culture at low temperature, but the total cell count after shifting the culture temperature to high temperature at time T1. The number and the number of plasmid-containing cells both increase remarkably. However, after time T2, the number of plasmid-containing cells is constant and does not increase (the line with black squares (■) in FIG. 1A). This is because the temperature-sensitive plasmid does not replicate at high temperatures, and the plasmid copy is distributed to daughter cells as the Escherichia coli grows, separation occurs, and a plurality of plasmid copies per cell are divided into daughter cells (partitioning). This is because, after a certain number of generations, after the time when the copy number per cell became 1 (T2), only Escherichia coli having no plasmid resulted in an increase in the number of cells. That is, cells containing the plasmid will be diluted in the total cell population. On the other hand, in low-temperature culture, the plasmid is replicated with the growth of Escherichia coli, and the total number of cells (大腸菌) and the number of Escherichia coli having the temperature-sensitive plasmid (●) increase with time.
[0013]
In the case of the ts-Seg plasmid whose separation is temperature-sensitive (FIG. 1B), all cells increase as plasmid-containing cells in low-temperature culture, but in culture after shifting the culture temperature to high temperature at time T1, After a certain period of time (T3-T1), the rate of increase in the total viable cell count is faster than the rate at low temperatures, as in the case of the ts-Rep plasmid, but T3-T1 is higher than T2-T1. In addition, the number of E. coli cells having the ts-Seg plasmid continues to increase after time T3, although the growth rate of the plasmid-containing cells is short (FIG. 1B, the line with black squares (■)). This means that at high temperatures, a copy of the ts-Seg plasmid is lost from daughter cells at a constant rate (30-35%) from generation to generation, and the plasmid carrying E. coli (FIG. 1B, line with black squares (■)) This is because the growth rate of Escherichia coli (the difference between the line marked with a square (□) and the line marked with a black square (■) in FIG. As a result, Escherichia coli having the ts-Seg plasmid is also diluted during growth at high temperatures, like E. coli having the ts-Rep plasmid.
The difference between time T2 and time T1 in FIG. 1A is the time required for the replication efficiency of the temperature-sensitive plasmid to decrease and for the appearance of cells without the ts-Rep plasmid due to separation. In the case of repAs1, the difference is calculated as the number of generations. 3.8 for repAs1h14 and 5.0 for repAs1h14. Similarly, the difference between time T3 and time T1 in FIG. 1B is the time until the appearance of Escherichia coli having no temperature-sensitive plasmid for isolation, and in the case of repAs5, 2.1 times in terms of the number of generations. there were.
[0014]
FIG. 1C shows the result of the present study on what kind of behavior is exhibited when such two types of temperature-sensitive mutations are simultaneously introduced into the repA gene.
Plasmid pTH14kssh (3343 bp) having these two types of temperature-sensitive mutation and high copy number mutation at the same time was prepared as an example of the plasmid of the present invention. The arrangement of this plasmid is shown in FIG. The entire nucleotide sequence is shown in SEQ ID NO: 6) in the sequence listing. This plasmid has a pSC101 DNA replication initiation factor gene (repA) encoding a pSC101 DNA replication initiation factor mutant (RepAssh) having three amino acid substitutions. The base sequence encoding this pSC101 DNA replication initiation factor mutant protein (RepAssh) consists of 948 bases, and the base sequence is shown in SEQ ID NO: 4 in the sequence listing. The amino acid sequence is composed of 316 amino acid residues, and the amino acid sequence is shown in SEQ ID NO: 2 in the sequence listing. FIGS. 3 and 4 show a comparison of the nucleotide sequences of this pSC101 DNA replication initiation factor mutant (RepAssh) and a wild-type pSC101 DNA replication initiation factor (RepAwt), and FIG. 5 shows a comparison of the amino acid sequences. As shown in FIGS. 3 and 4, the coding region of this pSC101 DNA replication initiation factor mutant gene (repAssh) has the seventh base guanine (G) mutated to adenine (A) (mutation at this site). Is called h14 mutation), guanine (G) which is the 37th base is mutated to adenine (A) (the mutation at this site is called s5 mutation), and cytosine (C) which is the 167th base Is mutated to thymine (T) (mutation at this site is called s1 mutation). In the amino acid sequence, as shown in FIG. 5, glutamic acid (E) as the third amino acid is substituted with lysine (K), alanine (A) as the 13th amino acid is substituted with threonine (T), The 56th amino acid, alanine (A), has been replaced with valine (V).
[0015]
For the following description, among the 316 amino acid residues of the protein encoded by the repA gene, a gene in which only s1 is mutated is referred to as “repAs1”, and a gene in which only s5 is mutated is referred to as “repAs5”. In addition, those in which only h14 is mutated are referred to as “repAh14” or the like. And the thing which mutated s1 and s5 is described as "repAss", and the thing which mutated three places of s1, s5, and h14 is described as "repAssh". In addition, each protein is represented by initial capital letters such as “RepAs1”.
The copy number of the pSC101 plasmid having repAs1 is "5" in terms of chromosome of E. coli host cells, which is not different from that of the wild type (repAwt), but a mutant that completely stops plasmid replication at high temperature. It is. repAs5 reduces the plasmid copy number from "5" to "1" at low temperatures, and at high temperatures during cell division, the transfer of plasmid copies to daughter cells becomes unstable. This is a mutant that loses and becomes plasmid-free. repAh14 was selected by treating a pSC101-based plasmid conjugated to an ampicillin resistance gene with hydroxyamine and increasing its drug resistance by about 4-fold, and increasing the plasmid copy number from "5" to "14" Mutant
[0016]
The coding region of the pSC101 DNA replication initiation factor mutant gene (repAsh) having these three mutations at the same time is inserted from the 1815th adenine base to the 2762nd cytosine base in the plasmid pTH14kssh (3343bp). This plasmid contains the resistance gene agp5 to confer resistance to the antibiotic kanamycin to the host bacterium. For insertion of a foreign gene, a single cleavage site of various restriction enzymes (NdeI, HindIII, SphI, PstI, SalI, XbaI, BamnHI, SmaI, KpnI, SacI, EcoRI) is replaced by a lac promoter operator sequence (POlac). It is located downstream. ATG in the NdeI recognition site (CATATG) is a translation initiation site of mRNA transcribed from POlac. Furthermore, a base sequence consisting of about 300 bases for adjusting the half-life of the mRNA of the pSC101 DNA replication initiation factor mutant gene (repAssh) is arranged downstream of the gene encoding the pSC101 DNA replication initiation factor mutant (RepAssh). ing.
[0017]
This plasmid is relatively stable in E. coli cells as a low copy number (≦ 1 / chromosome) plasmid at low temperatures (<30 ° C.) but grows as an independent plasmid molecule at high temperatures (≧ 37 ° C.) It was not maintained intracellularly. FIG. 1C shows the growth of Escherichia coli having this plasmid.
That is, at low temperatures, Escherichia coli grows while retaining the plasmid, but at high temperatures, replication of the plasmid does not occur at all, and Escherichia coli holding the plasmid does not grow. In addition, since the plasmid copy number is 1 or less per host chromosome even at a low temperature, Escherichia coli having the plasmid does not increase almost immediately after the culture temperature is shifted to a high temperature. Such behavior is very similar to the case of the temperature-sensitive plasmid for replication (see FIG. 1A), except that the difference between time T4 and time T1 is very short and both are almost simultaneous. . That is, by introducing both mutations at the same time, the growth of Escherichia coli having the plasmid is stopped almost simultaneously with the temperature shift-up or in a very short time.
As described above, the plasmid having the pSC101 DNA replication initiation factor mutant gene (repAssh) of the present invention has very sensitive temperature characteristics as compared with the conventional plasmid. By utilizing this phenomenon, the homologous recombination function can be compared with the wild-type (rec ⁺ LacI) and overexpresses the lactose promoter repressor ^q If the plasmid is introduced into a strain and cultured on an agar medium at a high temperature and a kanamycin-resistant colony is selected, a strain inserted into the chromosome by homologous recombination can be obtained. In this case, if a chromosomal fragment DNA of a non-essential region of Escherichia coli is previously inserted into the plasmid, the plasmid is inserted into that region (Hamilton et al., J. Bacteriol., 171: 4617-4622). , 1989). Further, by incorporating cDNA of a foreign gene downstream of POlac of the plasmid, the gene can be expressed as if it were an E. coli chromosome gene. In this case, lacI ^q Since the gene is present and only one copy of the gene per chromosome exists, it is possible to completely suppress the expression of the foreign gene. Moreover, this plasmid cannot replicate at 37 ° C., which is the optimum temperature for culturing Escherichia coli, and has excellent properties different from the conventional temperature-sensitive vector of repAs1 single mutation used by Hamilton et al. Thus, the plasmid is extremely useful as a foreign gene expression vector. In particular, as a host vector system for expressing a highly cytotoxic protein, a host cell capable of growing a host cell at 37 ° C. while suppressing the expression of a foreign gene, and having a gene encoding a highly cytotoxic protein Can be industrially grown in large quantities, and the expression of foreign genes can be switched only after IPTG is added after the cell concentration is sufficiently high. Therefore, even if the gene is highly cytotoxic or the host cell is killed by low-level expression, such "killing" can be avoided, and the target protein can be mass-produced. It becomes.
[0018]
The present invention has been described above using the plasmid having the repAssh gene as an example. The plasmid pTH14kshsh exemplified herein uses repAssh in which h14 is also mutated, but the mutation of h14 was used in the present invention for the purpose of convenience in plasmid construction, and is essential for the invention. It is not meant to be present, but for illustration. This is because it has been revealed in the course of our invention that the low copy number property of the s5 mutation is predominantly expressed in at least the cis relationship with respect to the high copy number property of the h14 mutation. In summary, it is important in the present invention that it is a double mutant of the s1 mutation and the s5 mutation, and the h14 mutation was only present to make the experiment easier.
That is, the present invention relates to a repA mutant having at least the s1 and s5 mutations (repAss: for example, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, or the base sequence represented by SEQ ID NO: 3 in the sequence listing) And a plasmid containing at least the mutant (for example, a plasmid pTH14kss having a base sequence represented by SEQ ID NO: 5 in the sequence listing). In another embodiment of the present invention, a repA mutant having an h14 mutation (repAssh: for example, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, or a base represented by SEQ ID NO: 4 in the sequence listing) A plasmid (for example, a plasmid pTH14kssh having a base sequence represented by SEQ ID NO: 6 in the sequence listing) containing the mutant.
Further, in another embodiment of the present invention, a single cleavage site (NdeI, HindIII, SphI, PstI) by various restriction enzymes for inserting a foreign gene downstream of the lac promoter / operator sequence (POlac) of the plasmid vector of the present invention. , SalI, XbaI, BamnHI, SmaI, KpnI, SacI, EcoRI) into which a high copy number plasmid such as pBR322 is inserted to form a composite plasmid. For example, a plasmid pTH14kssh: pBRH (7704 bp) can be obtained by inserting pBR322 (4361 bp) into the NdeI recognition site (CATATG) of the plasmid pTH14kssh of the present invention, and combining the plasmids pTH14kssh and pBR322. At this time, the insertion direction of pBR322 may be from any direction. For example, if the direction of DNA synthesis inserted in the same manner is “+” and the direction of DNA synthesis is “−”, the directions are expressed as pTH14kssh: pBRH + and pTH14kssh: pBRH−, respectively. These are convenient for preparing an expression vector for a foreign gene when constructing an expression vector by cutting out the pTH14kssh plasmid portion. The plasmid used at this time is not limited to pBR322 unless it is a pSC101 plasmid, but it is desirable that the size of the molecule is large enough to be separable from pTH14kss or pTH14kssh by a method such as electrophoresis. Similarly, it is also possible to insert pBR322 (4361 bp) into the NdeI cleavage site and the EcoRI cleavage site to obtain the composite plasmids pTH14kss: pBRN +, pTH14kss: pBRN-, pTH14kss: pBRE +, and pTH14kss: pBRE- (all 7704 bp). it can. Plasmids whose insertion direction is reversed are useful for different uses depending on the restriction enzyme sites in the nucleotide sequence of the foreign gene cDNA to be inserted downstream of POlac.
[0019]
The plasmid vector of the present invention is relatively stable in E. coli cells as a low copy number plasmid during growth at low temperatures (<30 ° C.), while being inserted into the chromosome during growth at high temperatures (> 37 ° C.). It is only maintained in cells in a state.
The plasmid vector of the present invention preferably has a gene that expresses resistance or immunity to an antibiotic or an antibacterial factor, and more preferably has an antibiotic resistance gene. In the example described in the sequence listing, a case using a kanamycin resistance gene is exemplified, but the present invention is not limited to kanamycin, and any other antibiotic resistance gene such as chloramphenicol and tetracycline may be used. Good.
Since the plasmid of the present invention has a low copy number, the DNA recovery rate may be extremely low even when cultured at 30 ° C. In such a case, an expression vector can be constructed by extracting a target plasmid DNA from a large-scale E. coli culture solution, but it is preferable to obtain DNA by performing a treatment for increasing the copy number. As a method of increasing the copy number for convenience, there is a method of preparing a composite plasmid with a high copy number plasmid as exemplified above.
[0020]
The plasmid of the present invention preferably has a promoter for expressing a foreign gene. Although various promoters can be used, the normally used lacUV5 promoter has a low level of expression even in the presence of a repressor, so that it is easier to switch on and off the wild-type lac promoter if possible. So preferred. However, it is not limited to these.
In addition, the plasmid vector of the present invention can insert a nucleotide sequence that regulates the half-life of mRNA into the downstream side of the repA gene, but is not limited thereto. It is preferable to use the original sequence of the repA gene as exemplified in the sequence listing as such a base sequence, but it is not limited thereto.
[0021]
The repA mutant gene of the present invention can be produced by various methods based on the nucleotide sequence disclosed by the present invention. For example, a pSC101-based plasmid can be mutated with hydroxyamine or another mutagen. As a result, it is possible to obtain those having the same effect as the s1 mutation or the s5 mutation, and those other than the s1 and s5 double mutants which cannot exist as a plasmid outside the chromosome at 35 ° C. or higher, like the s1 and s5 double mutants Wear. Alternatively, using any of these mutants as a template, a repA mutant gene having another mutation can be produced by a PCR method using primers having a different base. The h14 mutant can be produced by a similar method. Using the thus-produced RepA mutant gene (or repA mutant gene), a plasmid vector can be constructed according to a conventional method.
[0022]
A foreign gene can be inserted into the plasmid vector of the present invention according to a conventional method. Further, a plasmid into which a foreign gene has been inserted can also be inserted into the plasmid of the present invention. The foreign DNA may include not only the DNA of the gene to be expressed but also a homologous sequence of a non-essential region of Escherichia coli. This is because it will increase the frequency of insertion of the expression vector into the chromosome.
The plasmid vector of the present invention is characterized in that it contains a double mutant repA gene having at least a temperature-sensitive mutation s1 for replication and a temperature-sensitive mutation s5 for isolation, and the replication and isolation are performed at temperature. It is characterized in that it is sensitive and consequently cannot be maintained as an extrachromosomal plasmid in E. coli cells at a normal temperature (37 ° C.) or higher. Therefore, the other points can be handled in the same manner as a normal plasmid vector, preferably a pSC101 plasmid vector.
Escherichia coli is preferred as the host cell, but is not limited thereto.
[0023]
The temperature-sensitive plasmid vector of the present invention is a growing Escherichia coli that is relatively stable in cells as an extrachromosomal low copy number plasmid at low temperatures (<30 ° C.) and not so at high temperatures (> 37 ° C.). It is of a nature that can be replicated only as a part of the chromosome. Therefore, when an arbitrary gene is desired to be expressed using a coding region having an NdeI cleavage site upstream of a cDNA fragment and an EcoRI cleavage site downstream, the usual steps are as follows.
(1) DNA of the pTH14kssh: pBRN + complex plasmid is extracted from a small amount of E. coli culture (usually several ml) containing the plasmid, cut with NdeI and EcoRI, and electrophoresed on an agarose gel.
(2) Four types of DNA bands of about 3.3 kbp, 2.1 kbp, 1.3 kbp and 70 bp are obtained, respectively. However, those having a size of 70 bp are usually not observed visually. Of these, the band of 3.3 kbp belongs to the pTH14kssh plasmid itself, so this band is recovered and purified.
(3) The cDNA of the NdeI-EcoRI digestion fragment prepared in advance is enzymatically ligated to the above DNA. In this case, a DNA fragment having a length of about several hundred bp having homology to a non-essential region of E. coli chromosome is inserted downstream of the 3'-untranslated region of the cDNA fragment. This determines the site of integration into the chromosome.
(4) Using the DNA ligated in (3), E. coli (rec ⁺ , LacI ^q (Preferably a strain), and a nutrient liquid medium is added thereto, followed by shaking at 30 ° C. for 2 hours or more.
(5) The above culture solution is appropriately diluted and spread on two nutrient agar media containing kanamycin (10 μg / ml).
(6) Incubate one of the two sets of agar medium at 28-30 ° C and the other at 40-42 ° C for 1-2 nights.
(7) 10 μg / μg of pTH14ksshDNA at 30 ° C. ⁴ -10 ⁷ It is confirmed that at least 42 transformants have been obtained, and at 42 ° C., it is confirmed that colonies smaller by several orders of magnitude have been obtained. If foreign DNA previously containing non-essential regions of the E. coli chromosome was inserted, the number of colonies formed at high temperatures would increase.
(8) The above colony obtained at 42 ° C. is cultured at 37 ° C. in a liquid medium to produce a seed culture, and stored in liquid nitrogen by a conventional method.
(9) A part of the above-mentioned seed culture is taken out and cultured in a liquid medium containing kanamycin at 37 ° C. or more, and it is confirmed that mRNA is synthesized from the foreign gene cDNA, and at least several ml of plasmid DNA is cultured. Make sure it is not recovered from the liquid.
(10) After confirming the above, a culture solution appropriately scaled up from the same badge seed culture is produced under the same conditions, and the production of the protein is confirmed by immunological or biochemical methods.
Since the plasmid of the present invention is a low copy number plasmid, the copy number of the plasmid may be extremely low in some cases.For example, the plasmid number may be 0.1 or less per cell. If handling becomes difficult in such a case, it is preferable to take measures to increase the copy number.
[0024]
【Example】
Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.
[0025]
Example 1 (Construction of plasmid pTH14kssh)
Each of the s1 mutation and the s5 mutation of the repA gene of the pSC101 plasmid has already been reported (Hashimoto-Gotoh and Sekiguchi, J. Bacteriol., 131, 405-412, 1977). Further, a three-drug resistant plasmid vector of ampicillin, chloramphenicol, and kanamycin, pHSG415s, which is temperature-sensitive for DNA synthesis using the repAs1 mutant, has already been reported (Hashimoto-Gotoh et al., Gene, 16, 227-235, 1981.).
(1) DNA of pHSG415s is randomly mutated with hydroxyamine by the method of Hashimoto and Sekiguchi (J. Bacteriol., 127: 1561-1563, 1976). Escherichia coli is transformed with the DNA mixture, shake-cultured in a nutrient medium at 30 ° C. for 2 hours, then added with 500 μg / m of ampicillin, and then cultured overnight under the same conditions. The next day, 0.5 ml of the mixture is transferred to a fresh nutrient medium containing the same ampicillin, and cultured for another 5 hours. This is repeated five times, and five single colonies are randomly selected. Each is cultured overnight at 30 ° C. in a liquid nutrient medium containing 50 μg / ml ampicillin. At this time, as a control, Escherichia coli having the original pHSG415s not treated with hydroxyamine is also cultured under the same conditions. The next day, add 10 ml of each culture. ^-6 A double-diluted solution is prepared, and 50 μl of the solution is spread on an agar medium containing 50 μg / ml, 100 μg / ml, 200 μg / ml, 400 μg / ml, and 800 μg / ml of ampicillin. After culturing at 30 ° C. for 2 nights, about 400 to 500 colonies were formed in the other five samples up to 400 μg / ml, while E. coli having the pHSG415s control formed colonies only up to 100 μg / ml. A plasmid DNA was extracted from one of these and named pHSG415sh.
[0026]
(2) A DNA fragment containing the repA gene and flanked by restriction enzymes HaeII-Ndel was prepared from the above-described pHSG415sh plasmid, and kanamycin resistance gene agp5 and NdeI, HindIII, SphI, PstI, Sa1I, XbaI, A vector having polycloning sites of BamHI, SmaI, KpnI, SacI, and EcoRI was constructed in the same manner as in the method of Hashimoto et al. (Hashimoto-Gotoh et al., Gene, 241, 185-195, 2000) and named pTH19ksh.
(3) PCR primers were designed as follows to introduce a mutation in repAs5 into pTH19ksh.
Forward primer (US5)
AAAATGAACTAACGATTAGTCGC
This is a primer for introducing the s5 mutation, and the underlined portion is G in the wild type.
Reverse primer (lacFW)
CGTTGTAAAACGACGGCCAG
This is the reverse primer for the repA gene designed for the PCR reaction.
DNA was amplified using these primers by a conventional method, and an attempt was made to construct a target plasmid. Kanamycin-resistant colonies obtained by transforming Escherichia coli (TOP10) using the PCR mixture DNA can be confirmed by a stereoscopic microscope or a loupe (diameter of 0.1 to 0.1%) even when cultured at low temperature for 2 days. 0.2 mm) was observed, and a normal size (> 1 mm) was not obtained. In addition, there was no change in the size of the colony even when left at 30 ° C. for about 4 nights.
This is because the repA gene in which the s1 mutation and the s5 mutation coexist becomes very unstable or inactive due to the presence of these double temperature-sensitive mutations in the RepA protein, and as a result, in the medium containing kanamycin. It was suggested that the host bacteria had become lethal.
[0027]
Example 2 (Construction of plasmid pTH14kssh)
The repA gene of pTH19ksh described in Example 1 has a deletion of about 300 bp from the single NdeI site downstream of the coding region to the 3 'region. Because of this, the mRNA of the transcript repA was unstable, and as a result, the activity of the RepA protein was quantitatively reduced, so that the DNA synthesis initiation activity could not be sufficiently exhibited. By restoring this region, it was verified whether the s1 mutation and the s5 mutation can coexist in the same gene. After cutting the NdeI site of pHSG415sh, it was blunt-ended with T4 DNA polymerase and religated, cut out as an Asel-AflIII fragment, and ligated to the AflIII site in the repA gene of pTH19ks1 and the Asel site at the upstream end of POlac to obtain pTH14ksh. STH mutation was introduced into the pTH14ksh DNA in the same manner as in Example 1 (2), and when transformed at 30 ° C., several hundred colonies were obtained on an agar medium containing kanamycin (15 μg / ml). After culturing at 30 ° C. for two nights, the obtained colonies had diameters of about 1.5 mm and about 2.0 mm at a ratio of about half. Plasmid DNA was extracted from each representative five colonies and the following primers (U1545 and L2038)
U1545 primer
5'-GAGTTATACACAGGGCTGGGG-3 '
L2038 primer
5'-AGTTCTCGTCATCAGGCTCTC-3 '
Was used to perform PCR amplification to determine the DNA base sequence, and it was confirmed whether mutations in s1, s5, and h14 had been introduced. All the mutations s1, s5, and h14 were present in the DNA obtained from the smaller colony, but only the s1, h14 mutation was present in the DNA obtained from the larger colony, and the s5 mutation was Had not been introduced. This indicates that when the 3'-region of the repA gene is restored, the RepAsh protein enables the growth of the host bacterium in the presence of the selected drug, kanamycin, even though the specific activity of the RepAsh protein is lower than that of the RepAsh protein. Suggested that the functions of plasmid replication and distribution work.
[0028]
The results of examining the characteristics of the obtained plasmid are shown in Table 1 below, in comparison with the wild type, s1 mutant type, and s5 mutant type.
[Table 1]

[0029]
In Table 1, "AA3" indicates the third amino acid from the N-terminus, "AA13" indicates the thirteenth amino acid from the N-terminus, and "AA56" indicates the 56th amino acid from the N-terminus. The “DNA replication efficiency” k is given by the equation
k = 2 · exp [ln (b _n / C _n ) / (N-n)]-1
(See Hashimoto-Gotoh et al., Gene, 241, P185-191 for each parameter), and the values in the table are relative DNA at high temperature (42 ° C) and low temperature (30 ° C). It is expressed as replication efficiency. The growth rate of the host cell containing the plasmid in FIG. 1 corresponds to the value obtained by dividing the slope of the straight line on the high temperature side by the slope of the straight line on the low temperature side. The “colony forming ability” is a value obtained by dividing the number of colonies formed by the plasmid-carrying bacteria at 42 ° C. by the number of colonies formed at 30 ° C. "Copy number" is the copy number of the plasmid per chromosome number of the host cell at low temperature (30 ° C). The underlined plasmid lacks the 3'-untranslated region which is presumed to regulate the half-life of the repA gene mRNA. In the case of pTH19kssh shown in parentheses, growth of kanamycin-resistant bacteria was hardly observed even at 30 ° C.
[0030]
【The invention's effect】
The plasmid vector of the present invention has a sensitive temperature sensitivity, and by changing the culture temperature, (1) plasmid replication does not occur during growth of the host bacterium at 42 ° C. No kanamycin-resistant colony-forming Escherichia coli host cells were present in which only the plasmid was integrated into the chromosome. (2) In such Escherichia coli, the lactose promoter on the plasmid was replaced by the lacI of the chromosomal gene. ^q (3) that the plasmid once integrated is stably transmitted to progeny cells as a part of chromosomal DNA without being cut off from the chromosome under culture conditions of 37 ° C. or higher, (4) By adding IPTG to a sufficiently grown cell culture to switch the lactose promoter, industrial production of a protein that is toxic to host cells can be efficiently performed. Escherichia coli used as a host is recA. ⁺ , OmpT ⁻ , LacI ^q , ΔlacU169. Each gene is integrated into the chromosome of the plasmid, the enzyme to decompose the recombinant protein, the lactose promoter is switched off, and the lactose promoter of the recombinant protein gene is added when IPTG is added. This is useful for preventing the switch from being turned on. Through these steps, it becomes possible to express a substance that is highly cytotoxic to cells, and industrial production of recombinant proteins by prokaryotic cells such as Escherichia coli is made possible.
[0031]
[Sequence list]

[Brief description of the drawings]
BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows the temperature of cells containing the temperature-sensitive plasmid pTH19ks1h14 for replication (FIG. 1A), the temperature-sensitive plasmid pTH19ks5 for isolation (FIG. 1B), and the plasmid pTH14kssh of the invention (FIG. 1C). The growth rates at 30 ° C.) (the circles and black circles in FIG. 1) and at high temperature (41.5 ° C.) (the squares and black squares in FIG. 1) were graphed. Things. In FIG. 1, the vertical axis indicates the logarithm of the number of bacteria / ml, and the horizontal axis indicates the culture time (hour). ○, total number of cells at 30 ° C .; ●, number of kanamycin-resistant cells at 30 ° C .; □, total number of cells at 42 ° C .; Δ, number of kanamycin-resistant cells at 42 ° C.
FIG. 2 shows an example of a plasmid vector of the present invention. s1, s5 and h14 indicate the mutation site of the present invention, and examples of the insertion site of pBR322 are shown. →, ← indicate the direction of DNA replication of pBR322, with + being the same direction as pTH14kssh and − being the opposite direction.
FIG. 3 shows a comparison between the nucleotide sequence of the pSC101 DNA replication initiation factor mutant (repAssh) and the nucleotide sequence of the wild-type (repAwt) in the plasmid vector of the present invention exemplified in FIG. It shows that there are mutations at the 7th, 37th and 167th bases.
FIG. 4 shows a comparison of the nucleotide sequence continued from FIG.
FIG. 5 shows a comparison between the amino acid sequence of pSC101 DNA replication initiation factor mutant protein (RepAssh) and the amino acid sequence of wild-type protein (RepAwt) in the plasmid vector of the present invention exemplified in FIG. is there. Indicates that there are substitutions at amino acids 3, 13, and 56.

Claims (25)

pSC101 DNA replication initiation factor (RepA), wherein the thirteenth amino acid in the amino acid sequence is mutated to threonine and the 56th amino acid is mutated to valine; A functional pSC101 DNA replication initiation factor (RepA) mutant. pSC101 DNA replication initiation factor (RepA), wherein the third amino acid in the amino acid sequence is mutated to lysine, the thirteenth amino acid is mutated to threonine, and the 56th amino acid is mutated to valine. PSC101 DNA replication initiation factor (RepA) mutant having temperature-sensitive replication ability and temperature-sensitive separation ability. The pSC101 DNA replication initiation factor (RepA) mutant according to claim 1, wherein the pSC101 DNA replication initiation factor (RepA) mutant has an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. The pSC101 DNA replication initiation factor (RepA) mutant according to claim 1 or 2, wherein the pSC101 DNA replication initiation factor (RepA) mutant has an amino acid sequence represented by SEQ ID NO: 2 in the sequence listing. A gene encoding pSC101 DNA replication initiation factor (RepA), wherein the 37th base of the translation region is mutated to A (adenine) and the 167th base is mutated to T (thymine). A gene encoding a pSC101 DNA replication initiation factor (RepA) mutant having temperature-sensitive replication ability and temperature-sensitive separation ability. A gene encoding pSC101 DNA replication initiation factor (RepA), wherein the seventh base in the translation region is mutated to A (adenine), the 37th base is mutated to A (adenine), and the 167th base A gene encoding a pSC101 DNA replication initiation factor (RepA) mutant having a temperature-sensitive replication ability and a temperature-sensitive separability, characterized in that T is mutated to T (thymine). The gene encoding the pSC101 DNA replication initiation factor (RepA) mutant according to claim 5, wherein the translation region of the gene encoding the pSC101 DNA replication initiation factor (RepA) mutant has the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing. . The pSC101 DNA replication initiation factor (RepA) mutant according to claim 5 or 6, wherein the translation region of the gene encoding the pSC101 DNA replication initiation factor (RepA) mutant has the nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing. The gene to do. A plasmid vector comprising a gene encoding the pSC101 DNA replication initiation factor (RepA) mutant according to any one of claims 5 to 8. 10. The plasmid vector according to claim 9, wherein the plasmid vector has a lac promoter, a promoter derived from a lac promoter containing lacUV5, tac, trc, or any other promoter whose expression can be controlled. The plasmid vector according to claim 9 or 10, wherein the plasmid vector has a gene that expresses resistance or immunity to an antibiotic or an antibacterial factor. The plasmid vector according to claim 11, wherein the drug of the drug resistance gene is kanamycin, chloramphenicol, ampicillin, or tetracycline. The plasmid vector according to any one of claims 9 to 12, wherein the plasmid vector has a restriction enzyme site into which another plasmid vector can be inserted. 14. The plasmid vector according to claim 13, wherein the other insertable plasmid vector has a high copy number. The plasmid vector according to claim 14, wherein the other plasmid vector capable of inserting a high copy number is pBR322. The plasmid vector has a nucleotide sequence that regulates the half-life of mRNA downstream of the gene encoding the pSC101 DNA replication initiation factor (RepA) mutant according to any one of claims 5 to 8. 16. The plasmid vector according to any one of 15. The plasmid vector according to any one of claims 9 to 16, wherein the plasmid vector has a nucleotide sequence represented by SEQ ID NO: 5 or 6 in the sequence listing. The plasmid vector according to any one of claims 9 to 17, further comprising a foreign gene inserted therein. 19. The plasmid vector according to claim 18, wherein the foreign gene can be expressed. A cell transformed with the plasmid vector according to any one of claims 9 to 19. 21. The cell according to claim 20, wherein the cell is an intestinal bacterium, particularly E. coli. The replication and isolation of the plasmid according to any one of claims 9 to 19 is performed at a temperature different from the culture temperature of the cells, and the replication and isolation as independent plasmid molecules are not substantially performed at the culture temperature of the cells. A method for culturing cells, characterized by the following. 23. The method according to claim 22, wherein the cells are enterobacteria, especially E. coli. A method for producing a protein encoded by a foreign gene by the method according to claim 22 or 23. 25. The method according to claim 24, wherein the temperature for replication and isolation of the plasmid according to any one of claims 9 to 19 is 28 to 33 ° C, and the culture temperature for expression of the foreign gene is 35 to 43 ° C. Method.

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