JP4555920B2 - Protein mass expression system by mRNA stability control - Google Patents

Description

細胞を、１％Bactopeptone、０．５％酵母エキス、０．５％ＮａＣｌ、及び０．１％グルコースを含有するＬブロス（ｐＨ７．０）中３０℃で培養した。適当な抗生物質を、プラスミド担持細胞を培養するために添加した。
cafA::cat突然変異株を、以下のように構築した。(T. Ogura, Kumamoto University School of Medicine, Kumamoto, Japanから得た) pUC9のHincII部位に挿入されたpACYC184の１．３ｋｂのHaeII断片を担持する、pXX557からの１．３ｋｂのBamHI-HindIII上のcat遺伝子カセットを、T4 DNAポリメラーゼの処理によりその付着末端を平滑末端にした後、pMEL1（２８）上のcafA遺伝子内のユニークBglII部位に挿入した。得られたプラスミドpMEL1-cafA::catから、cafA::catとその隣接領域を含む７．８ｋｂのSalI断片をアガロース・ゲル電気泳動後に単離した。大腸菌 (Escherichia coli) K-12 recD 突然変異体株FS1576（２９）を、上記の単離された線状DNAで形質転換させ、そして上記のcafA::catを含有線状DNAと染色体cafA領域の間の相同的組換えにより形成されたクロラムフェニコール耐性コロニーを単離した。このcafA::cat構築物を、P1ファージ仲介形質導入により使用された他の株に移した。この染色体のcafA::cat挿入をサザン・ハイブリダイゼーションにより確認した（データを示さず）。
【００１９】
細胞タンパク質の分析
リン酸ナトリウム・バッファー(50mM, pH 7.0)中に懸濁させた細胞を音波処理により破壊した。未破壊細胞を除去した後、細胞溶解物を、超遠心分離(100,000 x g, 30分、4℃)により分画した。上澄を可容性画分として集めた。沈殿を同一バッファー中に懸濁させ、そして膜画分として集めた。タンパク質をＳＤＳ−ポリアクリルアミド・ゲル電気泳動により分離し、そしてゲルをクマシー・ブリリアント・ブルーにより染色した。
【００２０】
全細胞 RNA の単離と mRNNA の分析
全細胞ＲＮＡを、以下のように、先に（２７）に記載されたように単離し、そして分析した。全細胞RNAを以下に記載する迅速法により単離した。１００μｌの大腸菌 (Escherichia coli)培養物を遠心分離し、そして氷冷TE (10mM Tris-HCl, pH8.0, 1mM EDTA)で洗浄した。細胞を、１００μｌのTE中に懸濁させ、そして等容量の平衡化フェノール／クロロホルム及び１／１０容量のローディング染料溶液（０．３６％のブロモフェノール・ブルー、０．３６％のキシレン・シアノール、５０％のグリセロール）とともに激しくボルテックスすることにより直接抽出した。遠心分離（10,000 x g, 10分）後、上層の一部を修飾アガロース・ゲル電気泳動上に直接適用した。抽出されたRNAを、ホルムアルデヒド−アガロース・ゲル電気泳動(6.5%ホルムアルデヒド、1xMOPSバッファー[20mM 3-(N-モルフォロノ)−プロパン・スルホン酸pH7.0、5mM 酢酸ナトリウム、1mM EDTA]、１％アガロース)により分離した。ゲルを１μg/mlの臭化エチジウム溶液で染色し、そしてその後、UV照射（２５４ｎｍ）下で写真撮影した。
【００２１】
ノーザン・ハイブリダイゼーションのために、RNAを、キャピラリー法により、正電荷をもつナイロン膜 (Hybond-N+, Amersham International plc., Buckinghamshire, England)上に移した。ハイブリダイゼーション・プローブとして、adhE遺伝子の一部を含有する2.1kbのHindIII断片とmreB遺伝子の一部を含有する0.6kbのpstI断片を使用した。gapA遺伝子のためには、DNA断片を、以下のプライマー・セット：
5’-GACTATCAAAGTAGGTATCAACGGT-3’ （配列番号２）及び
5’-GATGTGAGCGATCAGGTCCAGAACT-3’ （配列番号３）
を用いたポリメラーゼ・チェイン・リアクション（PCR）により増幅した。ノーザン・ハイブリダイゼーションをGene Image キット (Amersham)を使用して行った。
【００２２】
リファンピシン−追跡実験
対数増殖細胞を、１５０μg/mlのリファンピシンで処理して、RNAの新規合成を阻害し、そして所定の時間が経過した後に、全RNAを先に記載したように抽出し、そして分析する。mRNAの半減期を、NIH Imageソフトウェアを使用してそのバンド強度を計測することにより決定する。
【００２３】
adhE-lacZ 融合遺伝子の構築
プロモーター配列、5’-非翻訳領域、及びコーディング領域の最初の２７ｂｐを含むadhE遺伝子の１．３ｋｂ上流領域を、以下のプライマー・セット：
5’-GATGTGGCGAAGTTAACATGATGG-3’ （配列番号４）及び
5’-GCTCTACGAGTGCGTTAACTTACGCGAC-3’ （配列番号５）
｛プライマー中、HpaI部位（下線）がクローニングのために導入されている。｝を使用してPCRにより増幅した。増幅されたDNAを、HpaIで消化し、そしてアンピシリン耐性遺伝子を担持するミニ−Fプラスミド上でadhEの第9コドンとlacZの第6コドンにおいてlacZ遺伝子とフレームをあわせて連結した。得られたpALF1と名づけたプラスミドを、rng::cat突然変異体株及びそれらの親株内に導入した。AdhE-LacZ融合物の発現を、ＳＤＳ−ポリアクリルアミド・ゲル電気泳動により又はβ−ガラクトシダーゼ活性の計測により調べた。β−ガラクトシダーゼ活性を、Millarの方法により測定した（１６）。
【００２４】
実施例１： rng::cat 突然変異体における AdhE タンパク質の過剰生産
大腸菌 (Escherichia coli)のrng::cat突然変異体株を分析して、本発明者は、rng::cat突然変異体GM11が、rng ⁺ 親株MC1061と比較したとき、約１００ｋDaの分子量をもつ多量のタンパク質を産生することを発見した（図１参照）。rng::cat株GM11は、その野生型株MC1061よりも約5倍以上の量の１００ｋDaタンパク質を産生した。この１００ｋDaタンパク質のほとんどは、その膜画分中に回収された。但し、上記タンパク質のかなりの量がその可容性画分中に回収された。上記１００ｋDタンパク質の過剰生産は、無傷の遺伝子としてrng遺伝子だけを担持するプラスミドpMEL2（２４）が上記突然変異体株に導入されたときに、野生型に復帰した（図１参照）。これは、上記１００ｋDaタンパク質の過剰生産がrng遺伝子の機能の欠陥によるということを示している。
【００２５】
上記の現象は、rng::cat突然変異が上記MC1061株に導入されたとき、顕著であった。W3110株又はCSH26株においては、上記１００ｋDaタンパク質の過剰生産はMC1061株の場合におけるものよりもかなり低かった（図６参照）。それゆえ、この現象のさらなる分析を、MC1061株とそのrng::cat誘導体GM11株を主に用いて行った。
【００２６】
上記１００ｋDaタンパク質を同定するために、このタンパク質のN−末端アミノ酸配列決定を行った。上記１００ｋDaタンパク質のN-末端部分の１８のアミノ酸残基の配列は、AKGQSLQDPFLNALRRER（配列番号１）であることが判明し、そしてそれはAdhEタンパク質、すなわち、大腸菌 (Escherichia coli)マップ上の２８分にあるadhE遺伝子によりコードされる発酵的アルコール・デヒドロゲナーゼ (alcohol dehydrogenase)の配列と同一であった（７）。但し、そのN-末端メチオニン残基は除去されていた。AdhEタンパク質の計算分子量、99,995は、SDS-ポリアクリルアミド・ゲル電気泳動上の、上記過剰生産されたタンパク質の推定値に合致した。このAdhEタンパク質はスピロソーム (spirosome)といわれる大きな複合体を形成し、そして超遠心分離により沈殿物中に主に回収されるということが報告されている（１１, ８）。さらに、AdhE破壊株は、上記rng::cat突然変異が導入されたときにさえ、もはや上記１００ｋDaタンパク質を産生しなかった（データを示さず）。これらの結果から、本願発明者は、上記１００ｋDaタンパク質はAdhEであると結論した。
【００２７】
実施例２： AdhE タンパク質の発現に対する rne-1 突然変異の効果
rng遺伝子産物、RNaseGは、ｍRNAの分解及びｒRNAプロセッシングに関わる（１４）RNaseEと、高い配列ホモロジー（１４）及び遺伝的相互作用（２６）を示すので、AdhEの発現に対するRNaseEの突然変異の効果を調べた。図2に示すように、MC1061のrne-1 (RnaseE^ts) 誘導体であるCH1828は、制限温度（４３℃）及び許容温度（３０℃）においてAdhEタンパク質を過剰生産せず、上記現象はRNaseG−特異的であることを示した。
【００２８】
実施例３： rng::cat 突然変異における adhE ｍ RNA の安定性向上
rng遺伝子はRNaseGをコードするので、adhE ｍRNAに対するrng::cat突然変異の効果を、ノーザン・ハイブリダイゼーションにより調べた。図３（A）に示すように、rng::cat突然変異株GM11におけるadhE ｍRNAのレベルは、rng ⁺ 株MC1061におけるレベルより約６倍高かった。他方、上記遺伝子マップ上３９分にあり、かつ、解糖系内のグリセルアルデヒド3リン酸デヒドロゲナーゼをコードするgapA遺伝子（１）のmRNAレベル、及び上記遺伝子マップ上rng遺伝子の直上流にあり、かつ、形態決定性タンパク質MreBをコードするmreB遺伝子（５）のmRNAレベルは、両株においてほぼ同じであった（図３（Ｂ）と３（Ｃ）参照）。
【００２９】
rng::cat突然変異がadhE遺伝子の転写又はadhE mRNAの安定性に影響を及ぼすかどうかを調べるために、リファンピシン (rifampicin)−追跡実験を行った。MRNAの新規合成は、１５０μg/mlのリファンピシンにより対数増殖細胞を処理することにより阻害され、そしてリファンピシン添加前に合成されたadhE mRNAの分解を、rng ⁺ 株とrng::cat株において調べた。図４（A）と４（C）に示すように、adhE mRNA は、rng ⁺ 細胞内において4.0分の半減期をもって分解された。一方、adhE mRNA は、rng::cat突然変異細胞内において9.7分の半減期をもって分解された。これは、rng::cat突然変異体株におけるAdhEタンパク質の過剰生産がadhE mRNAの安定性向上に因ることを意味する。adhE mRNA以外のmRNAの安定性に対するrng::cat突然変異の効果についても調べた。図４（B）と４（D）に示すように、gapA mRNAはrng ⁺ 株とrng::cat株において調べた。図４（A）と４（C）に示すように、adhE mRNA は両株においてほぼ同様の半減期をもって分解された； rng ⁺ 細胞内において7.9分、及びrng::cat突然変異細胞内において8.6分。これらは、rng遺伝子によりコードされるRNaseGがadhE mRNAを特異的に分解することを示唆している。但し、これは、RNaseGがadhE mRNA以外のmRNAを分解する可能性を排除するものではない。
【００３０】
実施例４： RNaseG −依存性の安定性制御における adhE mRNA の 5 ’ - 非翻訳領域の関わり
どんな因子がRNaseGによる安定性制御に関わっているかを調べるために、adhE-lacZ融合遺伝子を以下の実験手順に記載するように構築した。フレーム内にlacZ遺伝子と連結された、プロモーター配列、5’-非翻訳領域、及びコーディング領域の最初の２７ｂｐを含むadhE遺伝子の１．３ｋｂ上流領域を担持するプラスミドpALF1を、MC1061 (rng ⁺ )及びGM11 (rng::cat)内に導入した。図５（A）に示すように、AdhE-LacZ融合タンパク質は、染色体adhE遺伝子によりコードされる無傷のAdhEタンパク質とほぼ同じレベルまで、rng::cat突然変異体株GM11内において過剰生産された。GM11 (rng::cat)内のadhE-lacZ融合遺伝子から発現されたβ−ガラクトシダーゼ活性は、その親株MC1061 (rng ⁺ )における活性よりも約３．６倍高かった（図５（B）参照）。これは、adhE遺伝子のコーディング領域の大部分がRNaseGによる安定性制御のために必要でないということを意味している。
【００３１】
プラスミドALF1をCSH26 (rng ⁺ )及びそのrng::cat誘導体GC11内に導入した。面白いことに、AdhE-LacZ融合タンパク質の発現レベルは、MC1061におけるレベルよりも、rng ⁺ バックグラウンドにおいてさえ、CSH26においてかなり低かった（約１／５）。しかしながら、rng::cat突然変異の導入によるβ−ガラクトシダーゼ活性の上昇率は、CSH26とMC1061においてほぼ同じであった（図５（B）参照）。これは、adhE遺伝子の基礎的な転写レベルに影響を及ぼすであろういくつかの未知のトランス作用因子がMC1061とCSH26（及びたぶんW3110）の間で変わっているということ、及びこれがrng::cat突然変異の効果とは無関係であるということを、示唆する。
【００３２】
【発明の効果】
本願発明は、従来の転写制御による遺伝子発現調節に代わる、転写されたｍRNAの安定性制御による新規過剰発現系を提供する。本願発明に係る発現系においては、エネルギー分子（ヌクレオチド３リン酸）を消費する無駄な転写を最小限に抑えて目的のタンパク質を大量に生産することができる。
【００３３】

【００３４】
【配列表】

【図面の簡単な説明】
【図１】rng::cat突然変異株GM11におけるAdhEタンパク質の過剰発現を表す電気泳動ゲルの写真。MC1061 (rng ⁺)、GM11 (rng::cat)、及びGM11/pMEL2 (rng::cat/rng ⁺)の細胞タンパク質を可容性画分と膜画分に分画し、そして実験手順中に先に記載したように７．５％ＳＤＳ−ポリアクリルアミド・ゲル電気泳動上で分析した。クマシー・ブリリアント・ブルー染色ゲルを示す。ゲルの左に示す数字は、分子量（x kDa）である。
【図２】 AdhEタンパク質の発現に対するrne-1突然変異の効果を表す電気泳動ゲルの写真。３０℃で培養したMC1061 (rng ⁺)、GM11 (rng::cat)、及びCH1828 (rne-1)、並びに対数増殖期（ＯＤ６６０＝０．３）まで３０℃で培養し、そしさらに3時間４３℃にシフトさせたCH1828を、７．５％ＳＤＳ−ポリアクリルアミド・ゲル電気泳動上で分析した。クマシー・ブリリアント・ブルー染色ゲルを示す。ゲルの左に示す数字は、分子量（x kDa）である。
【図３】adhE、gapA、及びmreB mRNAに対するrng::cat突然変異の効果を表すノーザン・ハイブリダイゼーション結果。MC1061 (rng ⁺)及びGM11 (rng::cat)から抽出した全RNAを、実験手順中に記載したように、adhE（A）、gapA（B）、及びmreB（C）遺伝子のためのDNAプローブとのノーザン・ハイブリダイゼーションにより分析した。
【図４】リファンピシン−追跡実験の結果。MC1061 (rng ⁺)及びGM11 (rng::cat)の培養物を、RNAのde novo合成を阻害するために１５０μg/mlのリファンピシンで処理した。RNAを所定の時刻に抽出し、そして、図３におけるようにadhE（A）及びgapA（B）遺伝子のためのDNAプローブを用いたノーザン・ハイブリダイゼーションにより分析した。MRNAの半減期 (T _1/2)を、NIH Imageフトウェアを用いたバンド強度の計測により決定した。
【図５】adhE-lacZ融合遺伝子の発現に対するrng::cat突然変異の効果を表す電気泳動ゲルの写真である。adhE-lacZ融合遺伝子を担持するプラスミドALF1を、MC1061 (rng ⁺)、そのrng::cat誘導体GM11、又はCSH26 (rng ⁺)及びそのrng::cat誘導体GC11内に導入した。AdhE-LacZ融合タンパク質の発現を、７．５％ＳＤＳ−ポリアクリルアミド・ゲル電気泳動（A）により又はβ−ガラクトシダーゼ活性の計測（B）により調べた。平均±SD(n=3)を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for mass production of protein by improving the stability of mRNA of a target gene using a strain deficient in mRNA degrading enzyme, and an expression system used in the method. More particularly, the present invention relates to RNaseG-deficient strains,adhEThe present invention relates to a method for mass-expressing a protein by introducing a plasmid carrying a structural gene portion of a target gene connected downstream of the 5'-untranslated region, and an expression system used for the method.
[0002]
[Prior art]
RNaseG is an Escherichia coli (Escherichia coli) Is 71 minutes on the genetic maprngAn endoribonuclease responsible for 5'-end processing of the 16S rRNA encoded by the gene (13, 25, 27);cafAGene (E. coli (Escherichia coli) RNaseGrngThe former name of the gene was originally a putative open reading frame with an unknown function during the analysis of mecillinam-resistant mutants (orfF) (25). CafA protein overproduction is caused by E. coli (Escherichia coli) Caused the formation of linked cells and minicells. The inventor of the present application observes the fibrous structure along the long axis direction of the cell by observation with an electron microscope,caf (cytoplasmicaxialf(18). McDowall et al. (14) reported considerable sequence similarity between the CafA protein and the N-terminal half of RNaseE. RNaseE is an Escherichia coli (Escherichia coli) 24 minutes on the genetic maprneIt is encoded by a gene. RNaseE is an essential Escherichia coli (5) that is involved in 5S rRNA maturation and degradation of many mRNAs.Escherichia coli) Endoribonuclease (3).rne-1(ams-1Temperature sensitive mutations (also referred to as) were first described as increasing the chemical half-life of total RNA (12, 19). Other independently isolated mutations mapped as the same gene,rne-3071Leads to the accumulation of 9S rRNA, a precursor of 5S rRNA (6, 17). These two mutations were caused by amino acid substitutions within the highly homologous region between RNaseE and RNaseG (14).
[0003]
The inventor is directed to intracellular RNA metabolism.cafAInvestigate the effect of the mutation, and the precursor molecule of 16S rRNAcafA :: catIt was found to accumulate in the mutant. The inventor of the present invention has said that E. coli (BUMMER) accumulates a precursor of 16S rRNA called 16.3S rRNA having 66 extra nucleotides at its 5'-end.Escherichia coli) Mutant (4)cafAIt was also discovered that the gene has an 11 bp deletion (27). These results arecafAThe gene was shown to encode RNase activity involved in the 5'-terminal maturation of 16S rRNA defective in BUMMER mutants. The inventor has discovered that this activity is provided by a novel enzyme named RNaseG. This generngThe name was changed. Below, with CafAcafAInstead of RNaseG andrngIs used.
[0004]
The endoribonuclease activity of RNaseG was confirmed by Tock et al. Using a high purity protein and a synthetic oligoribonucleotide substrate in vitro (22). The function of RNaseG was dependent on the nature of the 5'-end of the substrate. RNaseG efficiently cleaves 5'-monophosphorylated substrates, but does not cleave 5'-hydroxylated substrates at all. The function of RNaseG is blocked by a 5'-triphosphate group. Interestingly, 5'-monophosphate groups can only stimulate cleavage at sites present on the same RNA molecule. In view of these characteristics, Tock et al., RNaseG completely eliminates 5'-monophosphorylated degradation or processing intermediates rather than initiating degradation of intact 5'-triphosphorylated RNA. Estimated to prefer cutting. In contrast, the 3'-phosphorylated state had no effect on the rate of cleavage by RNaseG. From the results of these in vitro experiments, Tock et al.Escherichia coli) Suggests that it has made some contribution to the degradation of RNA. The inventor's genetic evidence (26) showing that the effects of RNaseE and RNaseG are partially but overlapping also supports the estimation of Tock et al.
[0005]
By the way, the regulation mechanism of bacterial protein production, whether aerobic or anaerobic, is regulated at the transcription level such as promoter, operator, repressor, strength of SD sequence (ribosome binding sequence) and distance from translation start point. The general idea is that these are important factors. However, recently, it has been proposed that control by changing the stability of mRNA is important in order to respond quickly and appropriately to various environmental changes (30, 31). There is a lot of research on the mechanism of determination. Among them, RNaseE and RNaseIII which are site-specific endo-type RNases and PNPase which is an exo-type RNase are considered to play a particularly important role (17, 32, 33).
[0006]
In conventional protein expression systems, the target gene is placed under the control of a strong promoter, and the target protein is expressed in large quantities by increasing the synthesis of mRNA. Since the transcribed mRNA is rapidly degraded, synthesis must be performed at a rate exceeding the degradation in order to express a large amount of protein (increase the intracellular concentration of mRNA). Since the synthesis of mRNA is a process that consumes a large amount of nucleotide triphosphates (bioenergy molecules), the loss of energy is large in the expression method of the target protein using such a conventional expression system.
[0007]
In particular, E. coli (Escherichia coliIn the case of bacteria such as), energy cannot be produced by aerobic respiration (ATP) under anaerobic conditions, so that the energy is extremely limited compared to aerobic conditions. Therefore, in the conventional expression system using anaerobic bacteria, it was necessary to perform metabolism over a very long time. If a useful anaerobic reaction is discovered, it may be necessary to construct a mass expression system for polypeptides (eg, enzymes) involved in the reaction. However, the conventional method of linking the target gene to a strong promoter used under aerobic conditions cannot provide efficient expression because of insufficient energy supply. In particular, as described above, transcription consumes a large amount of nucleotide triphosphates (bioenergy molecules) (E. coli (Escherichia coli), About 20% of the cell weight is RNA molecules). Therefore, construction of an efficient energy-saving expression system is desired even under anaerobic conditions.
[0008]
[Problems to be solved by the invention]
Therefore, the problem to be solved by the present invention is to provide an expression method and an expression system that can achieve mass production of a target protein by minimizing energy loss by stabilizing mRNA.
[0009]
[Means for Solving the Problems]
The present inventor investigated whether RNaseG has an action on RNA molecules other than 16S rRNA, and as a result, for the first time, discovered that RNaseG degrades mRNA in vivo. And based on this discovery, the novel expression method and expression system for solving the said subject were constructed | assembled.
[0010]
In one embodiment of the present invention, a method for producing a protein by improving the stability of mRNA of a gene encoding the target protein, comprising the following steps:
Introducing an expression vector in which the gene encoding the target protein is operably linked to the 5′-untranslated region of the gene encoding the polypeptide overexpressed in the mRNA-degrading enzyme-deficient strain, This improves the stability of the transcribed mRNA;
Culturing the resulting strain; and
Recovering the target protein;
The production method is provided.
[0011]
In another aspect of the present invention, there is provided an expression system for a target protein:
an mRNA-degrading enzyme-deficient strain; and
An expression vector comprising:
A 5'-untranslated region of a gene encoding a polypeptide overexpressed by the defective strain; and
A gene encoding the target protein;
Including in an operable state;
Wherein the expression vector is introduced into the defective strain, thereby improving the stability of the mRNA of the gene encoding the target protein.
[0012]
The mRNA degrading enzyme may be RNaseG, the polypeptide overexpressed by the defective strain may be AdhE, and the defective strain may be E. coli (Escherichia coli) Derived from K-12 strain, such as GM11 (rng :: catAnd the protein of interest can be, for example, β-galactosidase, and the expression vector is operably further comprising a promoter sequence, such as pALF1.
Since the homologues of the RNaseG and RNaseE N-terminal endoribonucleotide degradation domains are present in most gram-negative and some gram-positive bacteria, these mRNA-degrading enzyme-deficient strains are those other than E. coli. You can also
[0013]
The mRNA-degrading enzyme-deficient strain GM11 (rng :: cat) And the polypeptide AdhE overexpressed by the deficient strain is preferred.
[0014]
Definition
As used herein, the term “polypeptide” also includes proteins and enzymes.
As used herein, the term “5′-untranslated region” means a part, not the whole, as long as it functions to increase the half-life of mRNA degradation.
As used herein, the term “mRNA stability” refers to an increase in the half-life of mRNA degradation, regardless of the mechanism of action of the mRNA-degrading enzyme.
[0015]
The present inventor examined whether RNaseG has an action on RNA molecules other than 16S rRNA. First, the present inventor found that a protein of 100 kDa was significantly overexpressed in the RNaseG-deficient strain. By protein N-terminal amino acid sequencing, the protein isadhEWas found to be an alcohol dehydrogenase encoded by (see Example 1). In order to clarify the level of regulation of AdhE overexpression (transcription or post-transcription), Northern hybridization was performed. In RNaseG-deficient strains,adhThe amount of E mRNA was increased about 6 times. Furthermore, by rifampicin chase experiment, in RNaseG deficient strain,adhE The half-life of mRNA was increased about 2.5 times (see Example 4). At this time, we examined as a controlgapAmRNA andmreB Since no such change was seen in mRNA, the action of RNaseG seems to be mediated by some recognition sequence. By RNaseGadhE The physiological significance of mRNA degradation is not yet clear, but it was transcribed in RNaseG-deficient strains.adhE Since mRNA exists stably, it can be said that alcohol dehydrogenase can be produced while minimizing wasteful transcription that consumes a large amount of bioenergy molecules.
[0016]
Next, RNaseG /adhETo see if the expression system of can be applied to other genes,adhEWhenlacZA fusion gene was constructed.adhEPromoter and 5'-untranslated regionlacZIn the gene-fused product, RNaseG deficiency caused a large amount of β-galactosidase expression at almost the same level as alcohol dehydrogenase (see Example 5). RNaseGadhE Cleavage of the 5'-untranslated region of mRNA causes degradation of the transcribed mRNA. In RNaseG-deficient strains, the transcribed mRNA accumulates stably in the cell without such cleavage. Therefore, it is considered that overexpression of alcohol dehydrogenase is caused. ThisadhEIt was proved that RNaseG-deficient strains can control expression by linking the 5'-untranslated region of this gene to the translated region of another gene.
[0017]
Hereinafter, the present invention will be described in detail with reference to examples. However, the technical scope of the present invention is not limited by the following examples.
[0018]
【Example】
Experimental procedure
Bacterial strains and media
E. coli used (Escherichia coliThe K-12 strains are listed in Table 1 below:
[Table 1]

The cells were cultured at 30 ° C. in L broth (pH 7.0) containing 1% Bactopeptone, 0.5% yeast extract, 0.5% NaCl, and 0.1% glucose. Appropriate antibiotics were added to culture plasmid-bearing cells.
cafA :: catMutant strains were constructed as follows. (Obtained from T. Ogura, Kumamoto University School of Medicine, Kumamoto, Japan) pUC9Hin1.3 kb of pACYC184 inserted into the cII siteHae1.3 kb from pXX557 carrying the II fragmentBamHI-Hinon dIIIcatAfter the gene cassette was blunted at its sticky end by treatment with T4 DNA polymerase, the gene cassette was transformed into pMEL1 (28).cafAUnique within a geneBglInserted at site II. The resulting plasmid pMEL1-cafA :: catFromcafA :: catAnd 7.8 kb including the adjacent areaSalThe I fragment was isolated after agarose gel electrophoresis. E. coli (Escherichia coli) K-12recD Mutant strain FS1576 (29) was transformed with the isolated linear DNA as described above andcafA :: catContains linear DNA and chromosomescafAChloramphenicol resistant colonies formed by homologous recombination between regions were isolated. thiscafA :: catThe construct was transferred to other strains used by P1 phage mediated transduction. Of this chromosomecafA :: catInsertion was confirmed by Southern hybridization (data not shown).
[0019]
Cellular protein analysis
Cells suspended in sodium phosphate buffer (50 mM, pH 7.0) were disrupted by sonication. After removing unbroken cells, the cell lysate was fractionated by ultracentrifugation (100,000 × g, 30 minutes, 4 ° C.). The supernatant was collected as an acceptable fraction. The precipitate was suspended in the same buffer and collected as a membrane fraction. Proteins were separated by SDS-polyacrylamide gel electrophoresis and the gel was stained with Coomassie Brilliant Blue.
[0020]
Whole cell RNA Isolation and mRNNA Analysis of
Total cellular RNA was isolated and analyzed as previously described in (27) as follows. Total cellular RNA was isolated by the rapid method described below. 100 μl of E. coli (Escherichia coliThe cultures were centrifuged and washed with ice cold TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). Cells are suspended in 100 μl TE and an equal volume of equilibrated phenol / chloroform and 1/10 volume of loading dye solution (0.36% bromophenol blue, 0.36% xylene cyanol, Extracted directly by vortexing vigorously (50% glycerol). After centrifugation (10,000 × g, 10 minutes), a portion of the upper layer was applied directly onto modified agarose gel electrophoresis. The extracted RNA was subjected to formaldehyde-agarose gel electrophoresis (6.5% formaldehyde, 1 × MOPS buffer [20 mM 3- (N-morpholono) -propane sulfonic acid pH 7.0, 5 mM sodium acetate, 1 mM EDTA], 1% agarose). Separated by. The gel was stained with 1 μg / ml ethidium bromide solution and then photographed under UV irradiation (254 nm).
[0021]
For Northern hybridization, RNA was transferred onto a positively charged nylon membrane (Hybond-N +, Amersham International plc., Buckinghamshire, England) by the capillary method. As a hybridization probe,adhE2.1kb containing part of the geneHindIII fragment andmreB0.6kb containing part of the genePSTI fragment was used.gapAFor genes, the DNA fragment is divided into the following primer sets:
5'-GACTATCAAAGTAGGTATCAACGGT-3 '(SEQ ID NO: 2) and
5'-GATGTGAGCGATCAGGTCCAGAACT-3 '(SEQ ID NO: 3)
Was amplified by polymerase chain reaction (PCR). Northern hybridization was performed using the Gene Image kit (Amersham).
[0022]
Rifampicin-chase experiment
Logarithmically growing cells are treated with 150 μg / ml rifampicin to inhibit RNA de novo synthesis, and after a predetermined time, total RNA is extracted and analyzed as described above. The half-life of mRNA is determined by measuring its band intensity using NIH Image software.
[0023]
adhE-lacZ Construction of fusion gene
Contains the promoter sequence, 5'-untranslated region, and the first 27 bp of the coding regionadhEThe 1.3 kb upstream region of the gene is represented by the following primer set:
5’-GATGTGGCGAAGTTAACATGATGG-3 '(SEQ ID NO: 4) and
5’-GCTCTACGAGTGCGTTAACTTACGCGAC-3 '(SEQ ID NO: 5)
{In primer,HpaThe I site (underlined) has been introduced for cloning. } Was used to amplify by PCR. The amplified DNAHpaOn a mini-F plasmid digested with I and carrying the ampicillin resistance geneadhE9th codon andlacZIn the 6th codon oflacZThe gene and frame were ligated together. The resulting plasmid named pALF1 wasrng :: catIt was introduced into mutant strains and their parental strains. Expression of the AdhE-LacZ fusion was examined by SDS-polyacrylamide gel electrophoresis or by measuring β-galactosidase activity. β-galactosidase activity was measured by the method of Millar (16).
[0024]
Example 1: rng :: cat In mutants AdhE Protein overproduction
E. coli (Escherichia coli)ofrng :: catAnalyzing the mutant strain, the inventorrng :: catMutant GM11 isrng ⁺ When compared to the parent strain MC1061, it was found to produce a large amount of protein with a molecular weight of about 100 kDa (see FIG. 1).rng :: catStrain GM11 produced about 100 times more protein than the wild-type strain MC1061. Most of this 100 kDa protein was recovered in the membrane fraction. However, a significant amount of the protein was recovered in the acceptable fraction. Overproduction of the above 100 kD protein is an intact generngWhen plasmid pMEL2 (24) carrying only the gene was introduced into the mutant strain, it reverted to the wild type (see FIG. 1). This is because overproduction of the 100 kDa proteinrngThis indicates that it is due to a defect in the function of the gene.
[0025]
The above phenomenonrng :: catThis was remarkable when the mutation was introduced into the MC1061 strain. In the W3110 strain or CSH26 strain, the overproduction of the 100 kDa protein was considerably lower than that in the case of the MC1061 strain (see FIG. 6). Therefore, a further analysis of this phenomenon is possible with the MC1061 strain and itsrng :: catDerivative GM11 strain was mainly used.
[0026]
In order to identify the 100 kDa protein, the N-terminal amino acid sequence of this protein was determined. The sequence of the 18 amino acid residues in the N-terminal part of the 100 kDa protein was found to be AKGQSLQDPFLNALRRER (SEQ ID NO: 1) and it was AdhE protein, ie E. coli (Escherichia coli) 28 minutes on the mapadhEIt was identical to the gene-encoded fermentative alcohol dehydrogenase sequence (7). However, the N-terminal methionine residue was removed. The calculated molecular weight of the AdhE protein, 99,995, matched the above estimate of overproduced protein on SDS-polyacrylamide gel electrophoresis. It has been reported that this AdhE protein forms a large complex called spirosome and is mainly recovered in the precipitate by ultracentrifugation (11, 8). In addition, the AdhE disruption strainrng :: catEven when the mutation was introduced, the 100 kDa protein was no longer produced (data not shown). From these results, the present inventors concluded that the 100 kDa protein is AdhE.
[0027]
Example 2: AdhE Against protein expression rne-1 Effect of mutation
rngThe gene product, RNaseG, shows high sequence homology (14) and genetic interaction (26) with RNaseE (14) involved in mRNA degradation and rRNA processing, so we investigated the effect of RNaseE mutation on AdhE expression It was. As shown in Figure 2, MC1061rne-1 (RnaseE^ts) The derivative CH1828 did not overproduce the AdhE protein at the limiting temperature (43 ° C.) and the permissible temperature (30 ° C.), indicating that the above phenomenon is RNaseG-specific.
[0028]
Example 3: rng :: cat In mutation adhE m RNA Improved stability
rngSince the gene encodes RNaseG,adhE against mRNArng :: catThe effect of the mutation was examined by Northern hybridization. As shown in FIG.rng :: catIn mutant strain GM11adhE The level of mRNA isrng ⁺ About 6 times higher than the level in strain MC1061. On the other hand, it is 39 minutes on the genetic map and encodes glyceraldehyde 3-phosphate dehydrogenase in the glycolysis systemgapAMRNA level of gene (1) and on the above gene maprngImmediately upstream of the gene and encodes the morphogenic protein MreBmreBThe mRNA level of gene (5) was almost the same in both strains (see FIGS. 3 (B) and 3 (C)).
[0029]
rng :: catMutationadhEGene transcription oradhE To determine if it affects mRNA stability, a rifampicin-follow-up experiment was performed. Novel synthesis of MRNA was inhibited by treating logarithmically growing cells with 150 μg / ml rifampicin and synthesized before the addition of rifampicinadhE mRNA degradation,rng ⁺ Stock andrng :: catInvestigated in stocks. As shown in FIGS. 4 (A) and 4 (C),adhE mRNA isrng ⁺ It was degraded intracellularly with a half-life of 4.0 minutes. on the other hand,adhE mRNA isrng :: catIt was degraded in the mutant cells with a half-life of 9.7 minutes. this is,rng :: catOverproduction of AdhE protein in mutant strainsadhE It means that the stability of mRNA is improved.adhE For the stability of mRNA other than mRNArng :: catThe effect of the mutation was also investigated. As shown in FIGS. 4 (B) and 4 (D),gapA mRNA isrng ⁺ Stock andrng :: catInvestigated in stocks. As shown in FIGS. 4 (A) and 4 (C),adhE mRNA was degraded with approximately the same half-life in both strains;rng ⁺ 7.9 minutes in the cell, andrng :: cat8.6 minutes in the mutant cell. They are,rngRNaseG encoded by the geneadhE This suggests that mRNA is specifically degraded. However, this is because RNaseGadhE It does not exclude the possibility of degrading mRNA other than mRNA.
[0030]
Example 4: RNaseG -In dependency stability control adhE mRNA of Five ' - Involvement of non-translated domains
To examine what factors are involved in stability control by RNaseG,adhE-lacZThe fusion gene was constructed as described in the experimental procedure below. In the framelacZContains the promoter sequence, 5'-untranslated region, and the first 27 bp of the coding region linked to the geneadhEPlasmid pALF1, carrying the 1.3 kb upstream region of the gene, was designated MC1061 (rng ⁺ ) And GM11 (rng :: cat). As shown in FIG. 5 (A), the AdhE-LacZ fusion protein is a chromosome.adhETo almost the same level as the intact AdhE protein encoded by the gene,rng :: catOverproduction in mutant strain GM11. GM11 (rng :: cat)InsideadhE-lacZΒ-galactosidase activity expressed from the fusion gene is expressed in its parent strain MC1061 (rng ⁺ ) Was about 3.6 times higher than the activity in) (see FIG. 5B). this is,adhEThis means that most of the coding region of the gene is not required for stability control by RNaseG.
[0031]
Plasmid ALF1 is converted to CSH26 (rng ⁺ ) And itsrng :: catThe derivative GC11 was introduced. Interestingly, the expression level of the AdhE-LacZ fusion protein is higher than the level in MC1061rng ⁺ Even in the background, it was fairly low (about 1/5) in CSH26. However,rng :: catThe rate of increase in β-galactosidase activity due to the introduction of the mutation was almost the same in CSH26 and MC1061 (see FIG. 5B). this is,adhEThat some unknown trans-acting factors that would affect the basic transcriptional level of the gene vary between MC1061 and CSH26 (and possibly W3110), and thisrng :: catIt suggests that it is unrelated to the effect of the mutation.
[0032]
【The invention's effect】
The present invention provides a novel overexpression system by controlling the stability of transcribed mRNA instead of the conventional regulation of gene expression by transcription control. In the expression system according to the present invention, the target protein can be produced in large quantities while minimizing wasteful transcription that consumes energy molecules (nucleotide triphosphates).
[0033]

[0034]
[Sequence Listing]

[Brief description of the drawings]
[Figure 1]rng :: catPhotograph of electrophoresis gel showing overexpression of AdhE protein in mutant strain GM11. MC1061 (rng ⁺), GM11 (rng :: cat), And GM11 / pMEL2 (rng :: cat / rng ⁺) Cell proteins were fractionated into a soluble fraction and a membrane fraction and analyzed on 7.5% SDS-polyacrylamide gel electrophoresis as previously described during the experimental procedure. Coomassie brilliant blue stained gel. The numbers shown on the left of the gel are molecular weights (x kDa).
[Figure 2] AdhE protein expressionrne-1A photograph of an electrophoresis gel showing the effect of the mutation. MC1061 cultured at 30 ℃ (rng ⁺), GM11 (rng :: cat), And CH1828 (rne-1), And CH1828 cultured at 30 ° C. until the logarithmic growth phase (OD660 = 0.3) and further shifted to 43 ° C. for 3 hours was analyzed on 7.5% SDS-polyacrylamide gel electrophoresis. Coomassie brilliant blue stained gel. The numbers shown on the left of the gel are molecular weights (x kDa).
[Fig. 3]adhE,gapA,as well asmreB against mRNArng :: catNorthern hybridization results showing the effect of the mutation. MC1061 (rng ⁺) And GM11 (rng :: cat), As described in the experimental procedure,adhE(A),gapA(B), andmreB(C) Analyzed by Northern hybridization with a DNA probe for the gene.
FIG. 4 Results of rifampicin-follow-up experiment. MC1061 (rng ⁺) And GM11 (rng :: cat) Cultures of RNAde novoTreated with 150 μg / ml rifampicin to inhibit synthesis. RNA is extracted at a given time and as in FIG.adhE(A) andgapA(B) Analysis was performed by Northern hybridization using a DNA probe for the gene. MRNA half-life (T_1/2) Was determined by measuring the band intensity using NIH Image software.
[Figure 5]adhE-lacZAgainst fusion gene expressionrng :: catIt is the photograph of the electrophoresis gel showing the effect of a mutation.adhE-lacZThe plasmid ALF1 carrying the fusion gene is called MC1061 (rng ⁺),Thatrng :: catDerivative GM11 or CSH26 (rng ⁺) And itsrng :: catThe derivative GC11 was introduced. Expression of the AdhE-LacZ fusion protein was examined by 7.5% SDS-polyacrylamide gel electrophoresis (A) or by measuring β-galactosidase activity (B). Mean ± SD (n = 3) is shown.

Claims (12)

A method of producing the protein by improving the stability of mRNA of a gene encoding the target protein, comprising the following steps: a polypeptide in which the defective strain is overexpressed in a strain deficient in RNaseG which is an mRNA degrading enzyme An expression vector in which a gene encoding the target protein is operably linked to the 5′-untranslated region of a gene encoding AdhE is introduced, thereby improving the stability of the transcribed mRNA; Culturing the obtained strain; and recovering the target protein. The method according to claim 1, wherein the defective strain is derived from Escherichia coli K-12. The method according to claim 2 , wherein the defective strain is GM11 ( rng :: cat ). The method according to any one of claims 1 to 3 , wherein the target protein is β-galactosidase. The method according to any one of claims 1 to 4 , further comprising a promoter sequence in a state in which the expression vector can act. 6. The method according to claim 5 , wherein the expression vector is pALF1. An expression system for the target protein: a RNaseG deficient strain that is an mRNA degrading enzyme; and an expression vector: a 5′-untranslated region of a gene encoding AdhE that is a polypeptide overexpressed by the deficient strain; Wherein the expression vector is introduced into the deficient strain, whereby the mRNA of the gene encoding the target protein is introduced. Said expression system, wherein stability is enhanced. The expression system according to claim 7, wherein the defective strain is derived from Escherichia coli K-12 strain. The expression system according to claim 8 , wherein the defective strain is GM11 ( rng :: cat ). The expression system according to any one of claims 7 to 9 , wherein the target protein is β-galactosidase. The expression system according to any one of claims 7 to 10 , further comprising a promoter sequence in a state where the expression vector can act. The expression system according to claim 11 , wherein the expression vector is pALF1.

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