JP4523660B2 - 発現ツールとしてのfkbpシャペロンの使用 - Google Patents
発現ツールとしてのfkbpシャペロンの使用 Download PDFInfo
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- JP4523660B2 JP4523660B2 JP2009025509A JP2009025509A JP4523660B2 JP 4523660 B2 JP4523660 B2 JP 4523660B2 JP 2009025509 A JP2009025509 A JP 2009025509A JP 2009025509 A JP2009025509 A JP 2009025509A JP 4523660 B2 JP4523660 B2 JP 4523660B2
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- Prior art keywords
- fusion protein
- protein
- chaperone
- fkpa
- polypeptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Description
〔1〕融合タンパク質を製造する方法であって、
a.標的ポリペプチドをコードする少なくとも1つのヌクレオチド配列およびSlyDであるFKBPシャペロンをコードする少なくとも1つのヌクレオチド配列をその上流に含んでなる、融合タンパク質をコードする組換えDNA分子であって、該融合タンパク質がシグナルペプチド配列を欠損しており、操作可能に連結されてなる組換えDNA分子を含んでなる発現ベクターを含む宿主細胞を培養する工程
b.該融合タンパク質の発現を誘導することにより、該融合タンパク質が封入体の形態で形成される工程
c.該融合タンパク質を該封入体から精製する工程
を含む方法、
〔2〕組換えDNAが標的ポリペプチドをコードする配列とFKBPシャペロンをコードする配列の間に位置された10〜100アミノ酸のペプチドリンカーをコードする少なくとも1つのヌクレオチド配列を含んでなることをさらに特徴とする〔1〕記載の方法、
〔3〕組換えDNA分子が、FKBPシャペロンをコードする1つのヌクレオチド配列を含んでなる、〔1〕又は〔2〕記載の方法、
〔4〕組換えDNA分子が、FKBPシャペロンをコードする2つのヌクレオチド配列を含んでなる、〔1〕又は〔2〕記載の方法、
〔5〕FKBPシャペロンをコードする2つの配列が標的ポリペプチドをコードする配列の上流に位置する、〔4〕記載の方法、
〔6〕FKBPシャペロンをコードする1つの配列が標的ポリペプチドをコードする配列の上流に位置し、FKBPシャペロンをコードするその他の配列が標的ポリペプチドをコードする配列の下流に位置する、〔4〕記載の方法、
〔7〕組換えDNA分子が10〜100アミノ酸のリンカーポリペプチドをコードする2つの核酸配列を含んでなる、〔4〕〜〔6〕いずれか記載の方法、
〔8〕〔1〕〜〔7〕いずれか記載の方法により得られる、組換えにより製造される融合タンパク質、
〔9〕研究動物の免疫化のための、〔8〕記載の組換えにより製造される融合タンパク質の使用、
〔10〕免疫学的検定法における〔8〕記載の組換えにより製造される融合タンパク質の使用
に関する。
第一の態様において、本発明は、標的ポリペプチドをコードする少なくとも1つのヌクレオチド配列およびFKBPシャペロンをコードする少なくとも1つのヌクレオチド配列をその上流に含む、融合タンパク質をコードする組換えDNA分子であって、該FKBPシャペロンがFkpA、SlyDおよびトリガー因子からなる群より選択されることに特徴を有する組換えDNA分子に関する。
本発明は、新規なポリペプチド発現系を記載する。好ましい態様において、標的ポリペプチドをコードする少なくとも1つのヌクレオチド配列およびFKBPシャペロンをコードする少なくとも1つのヌクレオチド配列をその上流に含む、融合タンパク質をコードする組換えDNA分子であって、該FKBPシャペロンがFkpA、SlyDおよびトリガー因子からなる群より選択されることに特徴を有する組換えDNA分子に関する。
s、Promega Biotechなどが挙げられるが、これらに限定されない)から得られ得る。さらに、構築物は、増幅可能な遺伝子(例えば、DHFE)に結合され得、その結果、遺伝子の複数のコピーが得られ得る。
1.1 FkpAおよびgp41を含む発現プラスミドの構築
野生型FkpAをクローニングし、発現させ、幾つかの重要でない改変をしたBothmannおよびPlueckthun, J Biol Chem 275 (2000) 17106-17113に従って精製した。貯蔵のため、タンパク質溶液を20mM NaH2PO4/NaOH(pH6.0)、100mM NaClに対して透析し、26mg/ml(1mM)に濃縮した。
発現プラスミドをもつE. coli BL21細胞を0.7のOD600に増殖させ、サイトゾル過剰発現を1mMのIPTGを37℃の増殖温度で添加することにより誘導した。誘導4時間後、細胞を遠心分離(5000gで20分)により収穫した。細菌ペレットを50mMリン酸ナトリウムpH7.8、6.0M GuHCl(塩化グアニジニウム)、5mMイミダゾールに再懸濁し、完全溶解のため室温で撹拌した(10分)。遠心分離(Sorvall SS34, 20000rpm,4℃)を繰り返した後、上清を濾過(0.8/0.2μm)し、溶解緩衝液中で予め平衡化したNi-NTAカラム(NTA:ニトリロトリアセテート;Qiagen;Germantown, MD)に適用した。非特異的に結合したタンパク質を、10カラム容積の溶解緩衝液を適用することにより洗浄工程中で除去した。最後に、結合標識タンパク質を、50mMリン酸ナトリウム、pH2.5、6.0M GuHClで溶出し、4ml画分に収集した。吸光度を280nmで記録した。
上記のように可溶化した物質を透析により生理的緩衝条件に移動する。透析チューブの選択カットオフ値は、4000〜6000ダルトンであった。
非折り畳みgp41-FkpAポリペプチド(50mMリン酸ナトリウム、pH7.8、7.0M GuHClに溶解)を、20mMリン酸ナトリウム、pH7.4、50mM NaCl、1mM EDTAで平衡化したSuperdex 200ゲル濾過カラムに適用した。FkpA-gp41は、本質的に3つの主な画分:高分子付随物として、明白なヘキサマー種として、および明白なトリマー種として溶出する。明白なトリマー画分を濃縮して、近UV CD測定におけるその三次構造について評価した(図4)。
シャペロンSlyDを、E. coliから慣用的なクローニング手順により単離している。組換え発現について、SlyDのアミノ酸1〜165をコードするDNA構築物を調製している。融合パートナーとしてSlyD(1〜165)および標的タンパク質としてHIV-1 gp41(配列番号:6を参照)を含む発現ベクターを構築している。融合タンパク質を発現し、上記FkpA-gp41について記載のように首尾よく精製した。興味深いことに、本発明者らは、SlyD(1〜165)-gp41型の天然様融合ポリペプチドが室温での50mMリン酸ナトリウムpH7.4、150mM NaClに対するカオトロピック物質(例えば、7.0M GuHClに溶解)の透析によりきわめて従来の様式で得られ得ることを見出した。
一本鎖PPIアーゼscSlyD(配列番号:7)およびscFkpA(配列番号:8)をそれぞれ、実施例1に記載されるのと実質的に同じ精製プロトコールに従ってE. coli過剰プロデューサーから得た。簡単には、誘導細胞を収穫し、PBSで洗浄し、50mMリン酸ナトリウムpH7.8、100mM塩化ナトリウム、7.0M GuHClで室温にて溶解した。非折り畳み標的タンパク質をそのC-末端ヘキサ-His-タグを介してNi-NTAカラムに結合し、50mMリン酸ナトリウムpH7.8、100mM塩化ナトリウム中で再折り畳みした。このマトリクス補助再折り畳み手順の後、タンパク質をイミダゾール勾配で溶出し、Superdex 200(登録商標)カラムでのゲル濾過に供した。
生化学的にすっかり異なる標的タンパク質HIV-1 gp41、HIV-2 gp36、HIV-1 p17およびHTLV gp21を、融合パートナー(gp41、gp36、p17、gp21)を有さないpET/BL21発現系を使用するか、または同じ標準的発現系であるが本発明の融合タンパク質(SlyD-gp41、FkpA-gp41、FkpA-p17、SlyD-gp36、FkpA-gp21)をコードするDNA構築物を含む系を使用するかのいずれかで発現している。これらの系の効率を、E. coli細胞質量あたりの組換えタンパク質の収率(mg/g)の点で比較している。表1から容易に明らかなように、新規な発現系は、試験した全てのタンパク質について有意な改善を導いた。
〔1〕 標的ポリペプチドをコードする少なくとも1つのヌクレオチド配列およびFKBPシャペロンをコードする少なくとも1つのヌクレオチド配列をその上流に含んでなる、融合タンパク質をコードする組換えDNA分子であって、該FKBPシャペロンがFkpA、SlyDおよびトリガー因子からなる群より選択されることに特徴を有する組換えDNA分子。
〔2〕 10〜100個のアミノ酸のペプチドリンカーをコードする少なくとも1つのヌクレオチド配列を、前記標的ポリペプチドをコードする配列と前記FKBPシャペロンをコードする配列との間に含むことにさらに特徴を有する前記〔1〕記載の組換えDNA分子。
〔3〕 前記FKBPシャペロンをコードするヌクレオチド配列を1つ含んでなる前記〔1〕または〔2〕記載の組換えDNA分子。
〔4〕 FKBPシャペロンをコードする配列を2つ含んでなる前記〔1〕または〔2〕記載の組換えDNA分子。
〔5〕 FKBPシャペロンをコードする2つの配列が標的ポリペプチドをコードする配列の上流に位置されることにさらに特徴を有する前記〔4〕記載の組換えDNA分子。
〔6〕 PPIシャペロンをコードする1つの配列が標的ポリペプチドの上流に位置され、PPIシャペロンをコードする他の配列が標的ペプチドをコードする配列の下流に位置されることにさらに特徴を有する前記〔4〕記載の組換えDNA分子。
〔7〕 10〜100個のアミノ酸のリンカーポリペプチドをコードする2つの核酸配列を含むことにさらに特徴を有する前記〔4〕〜〔6〕いずれか記載の組換えDNA分子。
〔8〕 10〜100個のアミノ酸のリンカーをコードする2つの核酸配列が異なる前記〔7〕記載の組換えDNA分子。
〔9〕 前記リンカー配列のうち少なくとも1つがタンパク質分解性切断部位を含むポリペプチドリンカーをコードする、前記〔2〕〜〔8〕いずれか記載の組換えDNA分子。
〔10〕 操作可能に連結された前記〔1〕〜〔9〕いずれか記載の組換えDNA分子を含んでなる発現ベクター。
〔11〕 前記〔10〕記載の発現ベクターで形質転換された宿主細胞。
〔12〕 a.前記〔11〕記載の宿主細胞を培養する工程
b.融合タンパク質の発現工程
c.該融合タンパク質の精製工程
を含む融合タンパク質を生成する方法。
〔13〕 FkpA、SlyDおよびトリガー因子からなる群より選択されるFKBPシャペロンに対応する少なくとも1つのポリペプチド配列ならびに標的ペプチドに対応する少なくとも1つのポリペプチド配列を含有してなる組換え的に生成された融合タンパク質。
〔14〕 FkpA、SlyDおよびトリガー因子からなる群より選択されるFKBPシャペロンに対応する少なくとも1つのポリペプチド配列、標的ペプチドに対応する少なくとも1つのポリペプチド配列、ならびに10〜100個のアミノ酸の少なくとも1つのペプチドリンカー配列を含有してなる組換え的に生成された融合タンパク質。
〔15〕 前記FKBPシャペロンに対応するポリペプチド配列を1つ含有することにさらに特徴を有する前記〔13〕または〔14〕記載の融合タンパク質。
〔16〕 前記FKBPシャペロンに対応するポリペプチド配列を2つ含有することにさらに特徴を有する前記〔13〕または〔14〕記載の融合タンパク質。
〔17〕 前記2つのFKBPシャペロンが標的ポリペプチドに関してN末端に位置されることにさらに特徴を有する前記〔16〕記載の融合タンパク質。
〔18〕 前記2つのFKBPシャペロンのうち1つが標的ポリペプチドに対してN末端に、前記FKBPシャペロンのうち1つが標的ポリペプチドに対してC末端に位置されることにさらに特徴を有する前記〔16〕記載の融合タンパク質。
〔19〕 少なくとも1つの標的ポリペプチド、FkpA、SlyDおよびトリガー因子からなる群より選択されるFKBPシャペロンに対応する2つの配列ならびに10〜100個のアミノ酸の2つのペプチドリンカー配列を含有してなる組換え的に生成された融合タンパク質。
〔20〕 前記ペプチドリンカー配列のうち少なくとも1つがタンパク質分解性切断部位を含有する、前記〔19〕記載の融合タンパク質。
〔21〕 前記標的タンパク質が感染性生物由来のポリペプチドを含有してなる前記〔13〕〜〔20〕いずれか記載の融合タンパク質。
〔22〕 前記ポリペプチドが感染性生物の少なくとも1つの診断的に直接関係のあるエピトープを含有することにさらに特徴を有する前記〔21〕記載の融合タンパク質。
〔23〕 研究動物の免疫化への、前記〔13〕〜〔22〕いずれか記載の組換え的に生成された融合タンパク質の使用。
〔24〕 ワクチンの生成における前記〔13〕〜〔22〕いずれか記載の組換え的に生成された融合タンパク質の使用。
〔25〕 免疫学的検定法における前記〔13〕〜〔22〕いずれか記載の組換え的に生成された融合タンパク質の使用。
〔26〕 前記〔13〕〜〔22〕いずれか記載の組換え的に生成された融合タンパク質、および薬学的に許容され得る賦形剤を含有してなる組成物。
Sambrook, J.ら,「Molecular Cloning: A LaboratoryManual」(1989)-, J. Sambrook,E.F. FritschおよびT. Maniatis編, Cold Spring Harbour LaboratoryPress, Cold Spring Harbour, NY
Beaucage, S.L.およびCaruthers, M.H., Tetrahedron Letters 22 (1981) 1859〜1862
Ausubel, F.ら,「Current protocolsin molecular biology」(1987および定期的改訂版), F. Ausubel, R. BrentおよびK.R.E.編, Wiley & Sons Verlag, New York
Fischer, G.ら, Nature 337 (1989) 476〜8
Hottenrott, S.ら, J Biol Chem 272 (1997) 15697〜701
Kapust, R.B.およびWaugh, D.S., Protein Sci 8 (1999) 1668〜74
Kay, J.E., Biochem J 314 (1996) 361〜85
Lane, W.S.ら, J Protein Chem 10 (1991) 151〜60
Matteucci, M.D.およびCaruthers, M.H., J. Am. Chem. Soc. 103 (1981) 3185〜3191
Metzger, D.ら, Nature 334 (1988) 31〜6
Rahfeld, J.U.ら, FEBS Lett 352 (1994) 180〜4
Ramm, K.およびPluckthun, A., J Biol Chem 275 (2000) 17106〜13
Schmid, F.X., Molecularchaperones in the life cyle of proteins (1998) 361〜389, A.L. FinkおよびY. Goto編, Marcel Decker In., New York
Scholz, C.ら, Embo J 16 (1997) 54〜8
欧州特許第293 249号
WO00/28011
WO93/25533
WO97/10253
WO98/1349
Claims (6)
- a.標的ポリペプチドをコードする少なくとも1つのヌクレオチド配列およびFKBPシャペロンをコードする1つのヌクレオチド配列をその上流に含んでなる融合タンパク質をコードする、操作可能に連結されてなる組換えDNA分子を含んでなる発現ベクターを含む宿主細胞を培養する工程、ここで、該融合タンパク質はシグナルペプチド配列を欠損している、
b.該融合タンパク質の発現を誘導することにより、該融合タンパク質が封入体の形態で産生される工程、および
c.該融合タンパク質を該封入体から精製する工程
を含む融合タンパク質を製造する方法であって、該FKBPシャペロンが大腸菌(E.coli) SlyDのアミノ酸1−165からなる、方法。 - 組換えDNAが、標的ポリペプチドをコードする配列とFKBPシャペロンをコードする配列の間に位置する10〜100アミノ酸のペプチドリンカーをコードする少なくとも1つのヌクレオチド配列を含んでなることをさらに特徴とする請求項1記載の方法。
- 組換えDNA分子が、10〜100アミノ酸のリンカーポリペプチドをコードする2つの核酸配列を含んでなる、請求項2記載の方法。
- 請求項1〜3いずれか記載の方法により得られる、組換えにより製造される融合タンパク質であって、該FKBPシャペロンが大腸菌(E.coli) SlyDのアミノ酸1−165からなる、融合タンパク質。
- 研究動物の免疫化のための、請求項4記載の組換えにより製造される融合タンパク質の使用。
- 免疫学的検定法における請求項4記載の組換えにより製造される融合タンパク質の使用。
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JP2003507262A Expired - Fee Related JP4262594B2 (ja) | 2001-06-22 | 2002-06-24 | レトロウイルス表面糖タンパク質を含む可溶性複合体 |
JP2006326846A Expired - Fee Related JP4309910B2 (ja) | 2001-06-22 | 2006-12-04 | 発現ツールとしてのfkbpシャペロンの使用 |
JP2007183508A Expired - Fee Related JP4320351B2 (ja) | 2001-06-22 | 2007-07-12 | レトロウイルス表面糖タンパク質を含む可溶性複合体 |
JP2008286959A Withdrawn JP2009102320A (ja) | 2001-06-22 | 2008-11-07 | レトロウイルス表面糖タンパク質を含む可溶性複合体 |
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EP (3) | EP1402041B1 (ja) |
JP (6) | JP4174422B2 (ja) |
KR (2) | KR100611545B1 (ja) |
CN (1) | CN100510068C (ja) |
AT (2) | ATE445016T1 (ja) |
AU (2) | AU2002317841A1 (ja) |
BR (1) | BRPI0210598B8 (ja) |
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