JP2020528760A - 新規融合タンパク質の調製およびそのタンパク質合成の向上における使用 - Google Patents
新規融合タンパク質の調製およびそのタンパク質合成の向上における使用 Download PDFInfo
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Abstract
Description
AはPabIエレメントであり、
Bは無いか、またはリンカーペプチドであり、
CはeIF4Gエレメントであり、
Sは任意のシグナルペプチドであり、
各「−」はペプチド結合である。)
(A)SEQ ID NO:3で表されるアミノ酸配列を有するポリペプチド、
(B)SEQ ID NO:3で表されるアミノ酸配列と80%以上の相同性(好ましくは、90%以上の相同性、より好ましくは、95%以上の相同性、最も好ましくは、97%以上の相同性、例えば、98%以上、99%以上)を有し、且つ、外来性タンパク質の発現効率を向上させる機能または活性を有するポリペプチド、
(C)SEQ ID NO:3のいずれかで表されるアミノ酸配列を、1〜15(好ましくは、2〜10、より好ましくは、3〜8)個のアミノ酸残基により置換、欠失、または付加することで形成され、外来性タンパク質の発現効率を向上させる機能または活性を有する誘導されたポリペプチド、
の群から選ばれる。
(a)外来性タンパク質の発現効率を向上させるという特性、
(b)インビトロ翻訳効率を向上させるという特性、
の群から選ばれる1つまたは複数を有する。
A1は、上記Aエレメントをコードするヌクレオチド配列であり、
C1は、上記Cエレメントをコードするヌクレオチド配列であり、
「−」は、エレメントA1とエレメントC1との間の結合手である。)
(i)(a)酵母細胞抽出物、(b)必要に応じるポリエチレングリコール、(c)必要に応じる外因性ショ糖、および(d)水または水性溶媒である必要に応じる溶媒を含む酵母によるインビトロタンパク質合成系と、
(ii)本発明の第1態様に記載の融合タンパク質と、
を含む外来性タンパク質を発現するためのインビトロタンパク質合成系を提供する。
(i)発現に適した条件で、本発明の第4態様に記載の宿主細胞を培養し、本発明の第1態様に記載の融合タンパク質を発現することと、
(ii)前記融合タンパク質を分離することと、
を含む本発明の第1態様に記載の融合タンパク質の生産方法を提供する。
(i)本発明の第1態様に記載の融合タンパク質を含む酵母によるインビトロタンパク質合成系を提供することと、
(ii)タンパク質の発現に適した条件で、前記外来性タンパク質のテンプレートの存在下で、前記酵母によるインビトロタンパク質合成系をインキュベートし、前記外来性タンパク質を発現することと、
を含む発現待ちの外来性タンパク質の発現方法を提供する。
本発明において、「Pab1」と「PabI」は、同じ意味を有し、交換使用可能なものである。
(a)酵母細胞抽出物と、
(b)ポリエチレングリコールと、
(c)必要に応じる外因性ショ糖と、
(d)水または水性溶媒である必要に応じる溶媒と、
を含むインビトロの無細胞のタンパク質合成系を提供する。
(i)酵母細胞を提供するステップと、
(ii)酵母細胞を洗浄処理し、洗浄された酵母細胞を取得するステップと、
(iii)洗浄された酵母細胞を細胞破砕処理し、酵母粗抽出物を取得するステップと、
(iv)前記酵母粗抽出物を固液分離し、液体部分、すなわち、酵母細胞抽出物を取得するステップと、
を含む。
(e1)RNAを合成するための基質、
(e2)タンパク質を合成するための基質、
(e3)マグネシウムイオン、
(e4)カリウムイオン、
(e5)緩衝剤、
(e6)RNAポリメラーゼ、
(e7)エネルギー再生システム、
の群から選ばれる1種または複数種の成分を含む。
(a)本発明は、初めて遺伝子組換え技術により、効率的な細胞形質転換ステージを介して、細胞内の遺伝子を組換え、翻訳システムのタンパク質の合成効率を向上させる。
(b)本発明は、インビトロタンパク質の合成能力を著しく強化できる融合タンパク質を初めて見出した。
(c)本発明は、eIF4Gの前に構成的または誘導性プロモーター(例えば、pScTEF1、pScPGK1、pKlTEF1、pKlPGK1、pScADH1、pScTPI1、pScTDH3、pKlADH1、pKlTPI1、pKlTDH3等)を挿入することにより、インビトロタンパク質の合成能力を著しく強化することができることを初めて見出した。
(d)本発明は、初めてCRISPR−Cas9遺伝子編集技術によりeIF4Gを組換え、インビトロタンパク質の合成能力を著しく強化する。
(e)本発明は、本発明の融合タンパク質がインビトロ翻訳システムの効率を向上させる際に、本発明の融合タンパク質のエレメントeIF4Gの発現量が増加しないことを初めて見出した。
ii.KleIF4G遺伝子開始コドンで、近接するPAM配列(NGG)を探索し、gRNA配列を確定した。GC含有量が適当であり、本発明の標準はGC含有量が40%〜60%であること、およびpoly T構造の存在を回避することをgRNA選択の原則とした。最終的には、本発明で確定した最適化されたKleIF4GのgRNA配列はCGGTTTTTCAAAGCAGATAT(SEQ ID NO.:6)であり、染色体Aの424927・・・・・・424936部位に位置する。
Claims (15)
- 式Iaまたは式Ibの構造を有する、ことを特徴とする融合タンパク質。
AはPabIエレメントであり、
Bは無いか、またはリンカーペプチドであり、
CはeIF4Gエレメントであり、
Sは任意のシグナルペプチドであり、
各「−」はペプチド結合である。) - 前記PabIエレメントAは、酵母のPabIタンパク質に由来する、ことを特徴とする請求項1に記載の融合タンパク質。
- 前記eIF4GエレメントCは、酵母のeIF4Gタンパク質に由来する、ことを特徴とする請求項1に記載の融合タンパク質。
- 請求項1に記載の融合タンパク質をコードする、ことを特徴とするポリヌクレオチド。
- 請求項4に記載のポリヌクレオチドを含む、ことを特徴とするベクター。
- 請求項5に記載のベクターを含むか、またはゲノムに請求項4に記載のポリヌクレオチドが組み込まれている、ことを特徴とする宿主細胞。
- (i)(a)酵母細胞抽出物、(b)必要に応じるポリエチレングリコール、(c)選択可能な外因性ショ糖、および(d)水または水性溶媒である選択可能な溶媒を含む酵母によるインビトロタンパク質合成系と、
(ii)請求項1に記載の融合タンパク質と、
を含む、ことを特徴とする外来性タンパク質を発現するためのインビトロタンパク質合成系。 - (iii)付加的に添加されたeIF4Gタンパク質を更に含む、ことを特徴とする請求項7に記載のインビトロタンパク質合成系。
- 前記eIF4Gタンパク質は、構成的または誘導性プロモーターにより誘導発現される、ことを特徴とする請求項8に記載のインビトロタンパク質合成系。
- (i)発現に適した条件で、請求項6に記載の宿主細胞を培養し、請求項1に記載の融合タンパク質を発現することと、
(ii)前記融合タンパク質を分離することと、
を含む、ことを特徴とする請求項1に記載の融合タンパク質の生産方法。 - インビトロタンパク質合成系のインビトロタンパク質の合成能力を向上させる製剤を調製するために用いられる、ことを特徴とする請求項1に記載の融合タンパク質の使用。
- (i)請求項1に記載の融合タンパク質を含む酵母によるインビトロタンパク質合成系を提供することと、
(ii)タンパク質の発現に適した条件で、外来性タンパク質のテンプレートの存在下で、前記酵母によるインビトロタンパク質合成系をインキュベートし、前記外来性タンパク質を発現することと、
を含む、ことを特徴とする発現待ちの外来性タンパク質の発現方法。 - 前記融合タンパク質は付加的に添加されたものである、ことを特徴とする請求項12に記載の方法。
- 前記ステップ(ii)は、外来性タンパク質活性の発現活性Q1を検出し、且つ、ステップ(ii)と同じ条件で野生型酵母株をインキュベートし、前記外来性タンパク質の活性Q2を検出し、Q1がQ2よりも著しく高ければ、外来性タンパク質の発現効率が著しく向上したことを示すというステップ(iii)を更に含む、ことを特徴とする請求項12に記載の方法。
- 外来性タンパク質を発現するインビトロタンパク質合成系を調製するために用いられ、前記インビトロタンパク質合成系は、外来性タンパク質の発現効率を向上させるために用いられる、ことを特徴とする請求項1に記載の融合タンパク質の使用。
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