JP4116054B2 - シャペロン融合タンパク質としての風疹e1エンベロープタンパク質変異体の組換え発現、可溶性で天然様かつ免疫反応性の抗原コンホメーションへのそのリフォールディングならびに抗風疹抗体の検出におけるその用途 - Google Patents
シャペロン融合タンパク質としての風疹e1エンベロープタンパク質変異体の組換え発現、可溶性で天然様かつ免疫反応性の抗原コンホメーションへのそのリフォールディングならびに抗風疹抗体の検出におけるその用途 Download PDFInfo
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Description
本発明の対象は可溶性風疹E1抗原及びこのペプチドの変異体であり、これらは、C末端における少なくとも膜貫通領域とアンカーセグメント、及び少なくともアミノ酸143-164を欠いていること、ならびに少なくともジスルフィド架橋Cys349-Cys352及びCys368-401にわたる領域を含有することにより特徴づけられ、一方、この領域のN末端(Cys349)は更に少なくとも15アミノ酸を含有し、及び/又は、この領域のC末端(Cys401)は更に隣接風疹E1抗原配列の少なくとも8アミノ酸を含有することにより特徴づけられる。ジスルフィド架橋Cys349-Cys352及びCys368-401にわたる領域は、この領域のN末端(Cys349)において、更に少なくとも隣接風疹E1抗原配列の25、30、34アミノ酸を含有し、及び/又は、この領域のC末端(Cys401)は更に隣接風疹E1抗原配列の少なくとも10、11、15、25、35アミノ酸を含有する。
本発明は、可溶性風疹E1抗原及びこのペプチドの変異体を開示する。これらは、C末端における少なくとも膜貫通領域とアンカーセグメント、及び少なくともアミノ酸143-164を欠いていること、ならびに少なくともジスルフィド架橋Cys349-Cys352及びCys368-401にわたる領域を含有することにより特徴づけられ、一方、この領域のN末端(Cys349)は更に少なくとも15アミノ酸を含有し、及び/又は、この領域のC末端(Cys401)は更に隣接風疹E1抗原配列の少なくとも8アミノ酸を含有することにより特徴づけられる。
a.宿主細胞の培養、
b.該融合タンパク質の発現、
c.該融合タンパク質の精製、
d.可溶性かつ免疫反応性のコンホメーションへのリフォールディング
の工程を含む。
Novagen(Madison, WI, USA)のpET24a発現プラスミドに基づき、以下のクローニング工程を行った。該ベクターをNdeI及びXhoIで消化し、縦列SlyD及び風疹E1断片201-432を含む半合成カセットを挿入した。この風疹E1断片はアミノ酸349、352、368及び401位にシステイン残基を含有する。
該発現プラスミドを含有する大腸菌(E. coli)BL21(DE3)細胞をLB培地+ カナマイシン(30μg/ml)内で、OD600が1になるまで増殖させ、37℃の増殖温度でイソプロピル-β-D-チオガラクトシド(IPTG)を最終濃度1mMまで加えることによりサイトゾル過剰発現を誘導した。誘導の4時間後、細胞を遠心分離(5000×gで20分間)し、凍結させ、-20℃で保存した。細胞溶解のために、該凍結ペレットを100mMリン酸ナトリウム(pH8.0)、7.0M GuHCl、10mM イミダゾールに室温で再懸濁させ、得られた懸濁液を2時間攪拌して細胞溶解を完了させた。遠心分離及び濾過の後、前記細胞溶解バッファー中で予め平衡化されたNi-NTA(ニッケル-ニトリロ-トリアセタート)カラム上にライセートをアプライした。未熟なジスルフィド架橋及びSSシャフリングを避けるために、金属キレート化カラムに適合しうる還元剤として5mM TCEPを洗浄バッファー中に加えた。過剰の洗浄工程(>20カラム容量の細胞溶解バッファー + TCEP)の後、マトリックス結合タンパク質のコンホメーションリフォールディングを誘導するために、該カオトロピック細胞溶解バッファーを50mMリン酸ナトリウム(pH7.8)、100mM塩化ナトリウム、5mM TCEPで置換した(残留GuHClがカオトロピック濃度で存在しないことが保証されるよう、少なくとも10カラム容量のリフォールディングバッファーをアプライした)。ついで、酸化的フォールディング(すなわち、システイン残基の酸化的架橋)を、50mMリン酸ナトリウム pH7,8、100mM塩化ナトリウムでの洗浄によりを誘導した。二価Ni2+イオンの有効濃度が高いため、マトリックス結合融合タンパク質内のジスルフィド架橋の形成は非常に速い過程である。溶出前に、〜50kDaの見掛け分子量を有する汚染(コンタミ)タンパク質を除去するために、該イミダゾール濃度を55mMまで上昇させた。ついで、50mMリン酸ナトリウム(pH7.8)、100mM塩化ナトリウム中の55mMから500mMまでのイミダゾール勾配を適用することにより、該天然融合タンパク質を溶出した。タンパク質含有画分を純度に関して評価し(SDS-PAGEによる判定で>95%)、プールした。最後に、該タンパク質をサイ
ズ排除クロマトグラフィーに付し、二量体画分をプールし、濃縮し、その分光学的特性に関して評価した。
a.UV分光学
SS-E1(201-432)は、可溶性かつ天然のようにフォールディングしたタンパク質として溶出する。組換え産生されマトリックスリフォールディングした融合タンパク質のUVスペクトルは凝集傾向を何ら示していない。図1に示すとおり、生理的バッファー条件中のSS-E1(201-432)のUV吸収スペクトルのベースラインは横軸にほぼ等しく(310nm以上)、したがってこのことは、自己会合又は凝集減少により生じる迷光粒子が存在しないことを示している。
融合ポリペプチド風疹SS-E1アミノ酸残基201-432の純度をSDS-PAGEにより確認した(図2)。2工程のクロマトグラフィーの後、該融合ポリペプチドの純度は95%を超えている。
その抗原性のほかに、SS-E1融合タンパク質のオリゴマー状態、溶解度及び安定性が診断目的に対するその適合性を決定する。該組換え風疹エクトドメイン断片のオリゴマー状態を明らかにするために、システイン含量において異なるSS-E1(201-432)をSuperdex 200HR 10/30カラム上の分析用ゲル濾過に付した。泳動バッファーは50mM リン酸ナトリウム(pH7.8)、100mM NaClであった。150μlのSS-E1(201-432)溶液(タンパク質濃度〜1.0mg/ml)をSECカラム上にアプライし、溶出を280nmの吸光度によりモニターした(図3)。該実験の結果は、すべてのSS-E1(201-432)システイン変異体が〜12.8mlで定量的に溶出したことを示しており、このことは見掛け上二量体の融合タンパク質を示している。これは、ジスルフィド結合の導入が風疹エクトドメインのポリペプチドバックボーンを拘束し該分子の全体的な小型化を招くという予想と一致する。FPLC分析における高分子会合の明らかな欠如はSDS-PAGEの結果を証明しており、SS-E1(201-432)のマトリックス共役酸化的リフォールディングが、相当に安定で異性化傾向もシャフリング傾向も示さない天然様ジスルフィド架橋を与えるという仮定を裏付けている。SS-E1(201-432)のオリゴマー化は、おそらく、安定な二量体を形成すると報告されている(Mukherjeeら, Biotechnol. Appl. Biochem. (2003) 37, 183-186; Mitterauerら, Biochem. J. (1999) 342, 33-39)シャペロン融合相手SlyDにより媒介されるのであろう。
組換え風疹エクトドメインのリシンε-アミノ基を〜10mg/mlのタンパク質濃度で、それぞれN-ヒドロキシ-スクシンイミド活性化ビオチン及びルテニウム標識により修飾した。標識/タンパク質のモル比は、それぞれの融合タンパク質に応じて2:1から5:1まで様々となった。反応バッファーは150mM リン酸ナトリウム(pH8.0)、50mM NaCl、1mM EDTAであった。反応を室温で15分間行い、緩衝化L-リシンを最終濃度10mMまで加えることにより停止させた。該カップリング反応の後、該粗タンパク質コンジュゲートをゲル濾過カラム(Superdex 200 HI Load)上に通過させることにより未反応遊離標識を除去した。
種々の該融合タンパク質の免疫反応性を自動Elecsys(登録商標)2010分析装置(Roche Diagnostics GmbH)において評価した。二重抗原サンドイッチ形態で測定を行った。それにより、該ビオチン-コンジュゲート(すなわち、捕捉抗原)をストレプトアビジンコート化磁気ビーズの表面上に固定化した。一方、検出抗原はシグナリング部分として錯化ルテニウムカチオンを含有する。Elecsys(登録商標)2010におけるシグナル検出は電気化学発光に基づくものである。特異的免疫グロブリンアナラアイトの存在下では、該発色性ルテニウム錯体は該固相へ架橋され、白金電極における励起の後に620nmの光を発する。ババリア赤十字の血清収集物からの抗風疹IgG陽性サンプルで測定を行った。
陽性として分類された全ての血清は、それで正しいと分析判定された。さらに、該結果は、陽性(COI* > 1)サンプルと陰性(COI* < 1)サンプルとの間の明らかな相違を示している。
システイン349と352との間、システイン349とシステイン352との間及びシステイン368と401との間でジスルフィド架橋を形成する風疹E1変異体の3つの異なる組合せの測定。前記風疹変異体と比較して、システイン非含有抗原を使用した。サンプル(WHO標準から)、及びババリア赤十字の血清収集物からの抗風疹IgG陽性サンプルを使用して、測定を行った。測定は、二重抗原サンドイッチ形態を用いて自動Elecsys(登録商標)2010分析装置(Roche Diagnostics GmbH)において行った。
ジスルフィド架橋の形成において異なる種々の変異体の比較は風疹SS-E1(aa315-432)の免疫反応性を示している。風疹SS-E1(aa315-432)システイン349とシステイン352との間及びシステイン368とシステイン401との間のジスルフィド架橋は適切に形成されている。陽性として分類された全ての血清は、それで正しいと分析判定された。さらに、該結果は、陽性(COI* > 1)サンプルと陰性(COI* < 1)サンプルとの間の明らかな相違を示している。
ババリア赤十字の血清収集物からの抗風疹IgG陽性サンプルを使用して、測定を行った。測定は、二重抗原サンドイッチ形態を用いて自動Elecsys(登録商標)2010分析装置(Roche Diagnostics GmbH)において行った。
「カットオフ」なる語は、陽性結果と陰性結果とを識別するためのシグナルである。すなわち、「シグナル > カットオフ」は陽性サンプルを表し、「シグナル < カットオフ」は陰性サンプルを表す。「COI」なる語はカットオフ指数として定義される。COIは、(サンプルのシグナル)/(カットオフでのシグナル)として計算される。すなわち、COI > 1は陽性サンプルを表し、COI < 1は陰性サンプルを表す。
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EP 0299673
EP 0061888
EP0944838
WO 03/000877
WO 98/13496
WO 03/000878
Claims (12)
- C末端における少なくとも膜貫通領域とアンカーセグメント、少なくともアミノ酸143-168、及びアミノ酸残基438-452の間のα-らせん領域を欠いていること、ならびに少なくともジスルフィド架橋Cys349-Cys352及びCys368-401を含む領域を含有することにより特徴づけられ、一方、この領域のN末端(Cys349)が更に隣接した風疹E1抗原配列の少なくとも34アミノ酸を含有し、かつ、この領域のC末端(Cys401)が更に隣接した風疹E1抗原配列の少なくとも11アミノ酸を含有することにより特徴づけられる可溶性風疹E1抗原であって、該風疹E1抗原がFKBPシャペロンに融合されている可溶性風疹E1抗原。
- 少なくともジスルフィド架橋Cys225-Cys235にわたる領域を更に含有することにより特徴づけられる、請求項1記載の可溶性風疹E1抗原。
- 該タンパク質が組換え融合タンパク質として製造される、請求項1又は2記載の可溶性風疹E1抗原。
- 請求項1〜3のいずれか1項記載の風疹E1抗原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り、その上流に、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が位置する、風疹E1抗原をコードする組換えDNA分子。
- 請求項4記載の組換えDNA分子を、機能しうる形で連結されて含んでなる発現ベクター。
- 請求項5記載の発現ベクターで形質転換された宿主細胞。
- 可溶性かつ免疫反応性の風疹E1抗原- FKBPシャペロン融合タンパク質の製造方法であって、
a.請求項6記載の宿主細胞の培養、
b.該融合タンパク質の発現、
c.該融合タンパク質の精製、
d.可溶性かつ免疫反応性のコンホメーションへのリフォールディング
の工程を含んでなる製造方法。 - サンプル中のIgG及び/又はIgMサブクラスの抗風疹抗体の検出及び測定及び定量のための方法であって、請求項1〜3のいずれか1項記載の風疹E1抗原を該抗体に対する捕捉試薬及び/又は結合相手として使用する、方法。
- 請求項1〜3のいずれか1項記載の第1風疹E1抗原と第2風疹E1抗原とを含んでなる、二重抗原架橋概念に従うイムノアッセイ。
- 請求項1〜3のいずれか1項記載の風疹E1抗原の少なくとも1つの抗原を含有する、抗風疹抗体の検出のための試薬キット。
- 風疹E1抗原のアミノ酸残基201-432、315-432、又は315-412の領域を含む、請求項1〜3のいずれか1項記載の可溶性風疹E1抗原。
- FKBPシャペロンがSlyDかFkpAである、請求項1〜3及び11のいずれか1項記載の可溶性風疹E1抗原。
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