JP5295242B2 - 細胞透過性Nm23組換えタンパク質、これをコードするポリヌクレオチド及びこれを有効成分として含有する癌転移抑制用組成物 - Google Patents
細胞透過性Nm23組換えタンパク質、これをコードするポリヌクレオチド及びこれを有効成分として含有する癌転移抑制用組成物Info
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Description
従って、本発明の目的は、癌転移抑制因子Nm23に細胞透過性を付与し、これを細胞内に高効率で導入することによって、様々な癌細胞で細胞増殖、分化及び移動の抑制を誘導し、優れた抗転移効果を奏することができる癌転移抑制剤としての細胞透過性Nm23組換えタンパク質を提供することである。
前記目的を達成するために、本発明は、癌転移抑制因子Nm23と巨大分子伝達ドメイン(MTD)の融合によって細胞透過性が付与され、Nm23を細胞内に高効率で導入する細胞透過性Nm23組換えタンパク質を提供する。
本発明の細胞透過性を有するNm23組換えタンパク質は、癌転移抑制因子であるNm23を細胞内に高効率で導入してKSRのリン酸化及び不活性化を誘導し、Ras−媒介MAPK連続段階を抑制することによって、癌細胞の増殖、分化及び移動を抑制し、癌転移を効果的に予防できる癌転移抑制剤として有用に用いられる。
[本発明101]
配列番号:2のアミノ酸配列を有するヒト癌転移抑制因子Nm23タンパク質のN末端、C末端、または両末端に、配列番号:4、6、8及び37〜227のアミノ酸配列からなる群から選ばれるアミノ酸配列を有する巨大分子伝達ドメイン(macromolecule transduction domain;MTD)が融合されていることを特徴とする、細胞透過性Nm23組換えタンパク質。
[本発明102]
前記巨大分子伝達ドメインが、配列番号:4のアミノ酸配列を有するkFGF4(kaposi fibroblast growth factor 4)由来MTD、配列番号:6のアミノ酸配列を有するJO-76MTD、及び配列番号:8のアミノ酸配列を有するJO-77MTDからなる群から選ばれることを特徴とする、本発明101の細胞透過性Nm23組換えタンパク質。
[本発明103]
前記組換えタンパク質の片末端に核局在配列(nuclear localization sequence;NLS)及びヒスチジンタグ親和性ドメインが共有結合されていることを特徴とする、本発明101の細胞透過性Nm23組換えタンパク質。
[本発明104]
下記からなる群から選ばれることを特徴とする、本発明101の組換えタンパク質:
配列番号:2のアミノ酸配列を有する全長Nm23のN末端に配列番号:4のアミノ酸配列を有するkFGF4由来MTDが融合された組換えタンパク質;
配列番号:2のアミノ酸配列を有する全長Nm23のC末端に配列番号:4のアミノ酸配列を有するkFGF4由来MTDが融合された組換えタンパク質;
配列番号:2のアミノ酸配列を有する全長Nm23の両末端に配列番号:4のアミノ酸配列を有するkFGF4由来MTDが融合された組換えタンパク質;
配列番号:2のアミノ酸配列を有する全長Nm23のN末端に配列番号:6のアミノ酸配列を有するJO-76MTDが融合された組換えタンパク質;
配列番号:2のアミノ酸配列を有する全長Nm23のC末端に配列番号:6のアミノ酸配列を有するJO-76MTDが融合された組換えタンパク質;
配列番号:2のアミノ酸配列を有する全長Nm23のN末端に配列番号:8のアミノ酸配列を有するJO-77MTDが融合された組換えタンパク質;
配列番号:2のアミノ酸配列を有する全長Nm23のC末端に配列番号:8のアミノ酸配列を有するJO-77MTDが融合された組換えタンパク質;
配列番号:2のアミノ酸配列を有する全長Nm23の両末端に配列番号:8のアミノ酸配列を有するJO-77MTDが融合された組換えタンパク質。
[本発明105]
配列番号:20、22、24、26、28、30、32、34及び36のアミノ酸配列からなる群から選ばれるアミノ酸配列を有することを特徴とする、本発明101の細胞透過性Nm23組換えタンパク質。
[本発明106]
本発明101の細胞透過性Nm23組換えタンパク質をコードするポリヌクレオチド。
[本発明107]
配列番号:19、21、23、25、27、29、31、33及び35の塩基配列からなる群から選ばれる塩基配列を有することを特徴とする、本発明106のポリヌクレオチド。
[本発明108]
本発明106のポリヌクレオチドを含む組換え発現ベクター。
[本発明109]
pET28a(+)-HM 1 N、pET28a(+)-HNM 1 、pET28a(+)-HM 1 NM 1 、pET28a(+)-HM 2 N、pET28a(+)-HNM 2 、pET28a(+)-HM 3 N、pET28a(+)-HNM 3 及びpET28a(+)-HM 3 NM 3 からなる群から選ばれることを特徴とする、本発明108の発現ベクター。
[本発明110]
本発明108の組換え発現ベクターで形質転換された形質転換細菌。
[本発明111]
大腸菌DH5α/HM 3 Nm23(KCTC-11380BP)であることを特徴とする、本発明110の形質転換細菌。
[本発明112]
大腸菌DH5α/HNm23M 3 (KCTC-11381BP)であることを特徴とする、本発明110の形質転換細菌。
[本発明113]
本発明110の形質転換細菌を培養する段階を含む細胞透過性Nm23組換えタンパク質を生産する方法。
[本発明114]
本発明101の細胞透過性Nm23組換えタンパク質を有効成分として含有し、薬学的に許容可能な担体を含む、癌細胞の増殖、分化または移動を遮断して転移を抑制するための癌転移抑制用薬学的組成物。
1)全長のNm23のN末端にkFGF4由来MTDが融合されたHis−MTD1−Nm23(HM1N)、
2)そのC末端にkFGF4由来MTDが融合されたHis−Nm23−MTD1(HNM1)、
3)その両末端にkFGF4由来MTDが融合されたHis−MTD1−Nm23−MTD1(HM1NM1)、
4)そのN末端にJO−76MTDが融合されたHis−MTD2−Nm23(HM2N)、
5)そのC末端にJO−76MTDが融合されたHis−Nm23−MTD2(HNM2)、
6)そのN末端にJO−77MTDが融合されたHis−MTD3−Nm23(HM3N)、
7)そのC末端にJO−77MTDが融合されたHis−Nm23−MTD3(HNM3)、
8)その両末端にJO−77MTDが融合されたHis−MTD3−Nm23−MTD3(HM3NM3)
である。
kFGF4由来MTD(MTD1)、JO−76MTD(MTD2)及びJO−77MTD(MTD3)のいずれか1つを巨大分子伝達ドメインとして用いて細胞透過性Nm23組換えタンパク質を製造するために、全長形態のNm23組換えタンパク質として下記8つの遺伝子コンストラクトを製造した(図1):
1)全長のNm23のN末端にkFGF4由来MTDが融合されたHis−MTD1−Nm23(HM1N)、
2)そのC末端にkFGF4由来MTDが融合されたHis−Nm23−MTD1(HNM1)、
3)その両末端にkFGF4由来MTDが融合されたHis−MTD1−Nm23−MTD1(HM1NM1)、
4)そのN末端にJO−76MTDが融合されたHis−MTD2−Nm23(HM2N)、
5)そのC末端にJO−76MTDが融合されたHis−Nm23−MTD2(HNM2)、
6)そのN末端にJO−77MTDが融合されたHis−MTD3−Nm23(HM3N)、
7)そのC末端にJO−77MTDが融合されたHis−Nm23−MTD3(HNM3)、及び
8)その両末端にJO−77MTDが融合されたHis−MTD3−Nm23−MTD3(HM3NM3)。
<2−1>最適な宿主菌株の選抜
細胞透過性Nm23組換えタンパク質の発現誘導に最も適した宿主菌株を選抜するために、LacIプロモーターを有する大腸菌BL21(DE3)、BL21 Gold(DE3)、BL21 CodonPlus(DE3)及びBL21 Gold(DE3)pLysS(Stratagene、USA)を対象に下記実験を行った。
前記実施例<2−1>で最適な菌株として確認された大腸菌BL21−Gold(DE3)に組換え発現ベクターpET28a(+)−HM1N、pET28a(+)−HNM1、pET28a(+)−HM1NM1、pET28a(+)−HM2N、pET28a(+)−HNM2、pET28a(+)−HM3N、pET28a(+)−HNM3及びpET28a(+)−HM3NM3それぞれを前記実施例<2−1>と同様の方法で形質転換させた後、0.7mMのIPTGを添加して発現を誘導し、これから得られた溶解性分画と不溶解性分画をそれぞれSDS−PAGEにかけてタンパク質発現特性と発現量の程度を分析した。
<3−1>組換えタンパク質の精製
本発明による細胞透過性Nm23組換えタンパク質は、封入体の形態で不溶解性分画に存在するので、これを精製するために、強力な変性剤として8M尿素を用いた。
前記実施例<3−1>のように、不溶解性分画から精製された本発明の組換えタンパク質は、強力な変性剤である8M尿素により変性されたため、これを活性形態に転換するために、下記の通りリフォールディング過程を行った。
本発明による細胞透過性Nm23組換えタンパク質の細胞透過性を定量的に検証するために、下記の通り、哺乳動物細胞におけるそれぞれの組換えタンパク質の細胞内流入を蛍光活性化細胞分取(fluorescence−activated cell sorting;FACS)で分析した。
細胞内に伝達された本発明による細胞透過性Nm23組換えタンパク質の細胞内位置を視覚的に確認するために、マウスの線維芽細胞に由来するNIH3T3細胞(韓国細胞株銀行、ソウル、大韓民国)をFITC標識された細胞透過性Nm23組換えタンパク質(HM1N、HNM1、HM1NM1、HM2N、HNM2、HM3N、HNM3及びHM3NM3)それぞれで処理した後、共焦点レーザー走査顕微鏡で観察した。
細胞透過性が立証されたNm23組換えタンパク質の細胞内機能を確認するために、3種類の癌細胞株を対象に前記組換えタンパク質の生化学的機能をウェスタンブロッティング分析で調査した。
<7−1>浸潤分析
本発明による細胞透過性Nm23組換えタンパク質で処理された癌細胞において、癌細胞の移動が遮断されて癌転移が抑制されるかどうかを確認するために、下記のような浸潤分析を行った。
本発明による細胞透過性Nm23組換えタンパク質が、移動能力に非常に優れた乳癌細胞株であるMDA−MB−435細胞の移動を抑制できるかどうかを確認するために、創傷移動分析(wound migration assay)を行った。
インビトロで立証された細胞透過性Nm23組換えタンパク質の転移抑制効果をインビボでも確認するために、下記免疫組織化学的分析(immunohistochemical analysis)を行った。
細胞透過性Nm23組換えタンパク質の投与後、腫瘍組織内でアポトーシスが誘導されるかを確認するために、前記実施例8のマウス動物モデルを用いてTUNEL分析を行った。この際、TUNEL分析は、in situ細胞死検出キット(in situ cell death detection kit)であるTMRレッド(TMR red、Roche)を用いた。
細胞透過性Nm23組換えタンパク質が投与された組織におけるタンパク質発現様相の変化を確認するために、下記の通りマイクロアレイ(microarray)分析を行った。
Claims (12)
- 配列番号:2のアミノ酸配列を有するヒト癌転移抑制因子Nm23タンパク質のN末端、C末端、または両末端に、配列番号:6及び8のアミノ酸配列からなる群から選ばれるアミノ酸配列を有する巨大分子伝達ドメイン(macromolecule transduction domain;MTD)が融合されていることを特徴とする、癌転移抑制または治療用細胞透過性Nm23組換えタンパク質。
- 前記組換えタンパク質の片末端に核局在配列(nuclear localization sequence;NLS)及びヒスチジンタグ親和性ドメインが共有結合されていることを特徴とする、請求項1記載の癌転移抑制または治療用細胞透過性Nm23組換えタンパク質。
- 下記からなる群から選ばれることを特徴とする、請求項1記載の組換えタンパク質:
配列番号:2のアミノ酸配列を有する全長Nm23のN末端に配列番号:6のアミノ酸配列を有する巨大分子伝達ドメイン(macromolecule transduction domain;MTD)が融合された組換えタンパク質;
配列番号:2のアミノ酸配列を有する全長Nm23のC末端に配列番号:6のアミノ酸配列を有する巨大分子伝達ドメイン(macromolecule transduction domain;MTD)が融合された組換えタンパク質;
配列番号:2のアミノ酸配列を有する全長Nm23のN末端に配列番号:8のアミノ酸配列を有する巨大分子伝達ドメイン(macromolecule transduction domain;MTD)が融合された組換えタンパク質;
配列番号:2のアミノ酸配列を有する全長Nm23のC末端に配列番号:8のアミノ酸配列を有する巨大分子伝達ドメイン(macromolecule transduction domain;MTD)が融合された組換えタンパク質;
配列番号:2のアミノ酸配列を有する全長Nm23の両末端に配列番号:8のアミノ酸配列を有する巨大分子伝達ドメイン(macromolecule transduction domain;MTD)が融合された組換えタンパク質。 - 配列番号:28、30、32、34及び36のアミノ酸配列からなる群から選ばれるアミノ酸配列を有することを特徴とする、癌転移抑制または治療用請求項1記載の細胞透過性Nm23組換えタンパク質。
- 請求項1記載の細胞透過性Nm23組換えタンパク質をコードするポリヌクレオチド。
- 配列番号:27、29、31、33及び35の塩基配列からなる群から選ばれる塩基配列を有することを特徴とする、請求項5記載のポリヌクレオチド。
- 請求項5記載のポリヌクレオチドを含む組換え発現ベクター。
- 請求項7記載の組換え発現ベクターで形質転換された形質転換細菌。
- 大腸菌DH5α/HM3Nm23(KCTC−11380BP)であることを特徴とする、請求項8記載の形質転換細菌。
- 大腸菌DH5α/HNm23M3(KCTC−11381BP)であることを特徴とする、請求項8記載の形質転換細菌。
- 請求項8記載の形質転換細菌を培養する段階を含む細胞透過性Nm23組換えタンパク質を生産する方法。
- 請求項1記載の細胞透過性Nm23組換えタンパク質を有効成分として含有し、薬学的に許容可能な担体を含む、癌細胞の増殖、分化または移動を遮断して転移を抑制するための癌転移抑制または治療用薬学的組成物。
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US96971407P | 2007-09-04 | 2007-09-04 | |
US60/969,714 | 2007-09-04 | ||
PCT/KR2008/005221 WO2009031835A2 (en) | 2007-09-04 | 2008-09-04 | Cell permeable nm23 recombinant proteins, polynucleotides encoding the same, and anti-metastatic composition comprising the same |
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US8168181B2 (en) | 2006-02-13 | 2012-05-01 | Alethia Biotherapeutics, Inc. | Methods of impairing osteoclast differentiation using antibodies that bind siglec-15 |
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EP3045532A1 (en) | 2006-06-02 | 2016-07-20 | President and Fellows of Harvard College | Protein surface remodeling |
JP2012525146A (ja) * | 2009-04-28 | 2012-10-22 | プレジデント アンド フェロウズ オブ ハーバード カレッジ | 細胞透過のための過剰に荷電されたタンパク質 |
KR101393397B1 (ko) * | 2011-11-23 | 2014-05-14 | 주식회사 프로셀제약 | 세포내 분자 전송 펩티드를 이용한 피부 생리 활성 분자의 경피 전달시스템 |
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US5807746A (en) * | 1994-06-13 | 1998-09-15 | Vanderbilt University | Method for importing biologically active molecules into cells |
US6495518B1 (en) * | 1994-06-13 | 2002-12-17 | Vanderbilt University | Method for importing biologically active molecules into cells |
US5874285A (en) * | 1996-09-13 | 1999-02-23 | Incyte Pharmaceuticals, Inc. | Polynucleotide encoding a novel human nm23-like protein |
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CN101918556A (zh) | 2010-12-15 |
EP2185707A2 (en) | 2010-05-19 |
EP2185707B1 (en) | 2013-07-03 |
KR20100076964A (ko) | 2010-07-06 |
KR101192860B1 (ko) | 2012-11-15 |
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