JP5288902B2 - キチン結合ドメインを用いる細胞表層提示方法 - Google Patents
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Description
分泌シグナル、キチン結合ドメイン、および提示されるべき該タンパク質をそれぞれコードするDNAを含む発現ユニットDNAを微生物に導入する工程;および
該発現ユニットDNAが導入された微生物を培養して、該タンパク質を該微生物の細胞表層に発現させる工程
を含む。
サッカロマイセス・セレビシエW303-1B株(アメリカンタイプカルチャーコレクション(ATCC)より入手)のゲノムDNAを鋳型として、配列表の配列番号2(forward)および配列番号3(reverse)に示されるプライマー対を用いてPCRを行うことにより、キチン結合ドメイン(CBD)を増幅した。
アスペルギルス・オリゼIF4 niaD-株の形質転換にpIS1-ss-N28-EGFPベクターを用いたこと以外は、実施例1に記載の方法と同様にして形質転換株を作製した。この形質転換により、EGFP分泌発現株を得た。
まず、実施例1のEGFP−CBD融合発現株および比較例1のEGFP分泌発現株のそれぞれの培養液からの細胞を超音波処理によって細胞破砕し、14000rpmにて4℃で5分間遠心分離し、培養上清画分、可溶性画分および細胞壁画分を得た。これらの画分を、一次抗体として抗EGFP抗体および二次抗体として抗マウスIgG抗体を用いるウェスタンブロッティングに供し、EGFP−CBD融合蛋白質の発現について分析した。対照として、アスペルギルス・オリゼIF4 niaD-未形質転換株もまた用いた。
実施例1のEGFP−CBD融合発現株および比較例1のEGFP分泌発現株、ならびに対照として用いたアスペルギルス・オリゼIF4 niaD-未形質転換株の培養液を、1×PBS溶液で30℃にて24時間処理した後、蛍光顕微鏡下で観察した。微分干渉顕微鏡下の観察との比較により、EGFP−CBD融合発現株では、細胞表層にEGFP由来の緑色蛍光が局在していることが分かった。EGFP分泌発現株および未形質転換株では、そのような蛍光分布は示さなかった。これにより、EGFP−CBD融合発現株では、EGFP−CBDが細胞表層に提示されていることが示された。
Claims (1)
- 糸状菌の細胞表層にタンパク質を提示させる方法であって、
分泌シグナル、キチン結合ドメイン、および提示されるべき該タンパク質をそれぞれコードするDNAを含む発現ユニットDNAを糸状菌に導入する工程;および
該発現ユニットDNAが導入された糸状菌を培養して、該タンパク質を該糸状菌の細胞表層に発現させる工程
を含み、
該キチン結合ドメインが、配列番号1に記載のアミノ酸配列を含むペプチド領域であり、
該タンパク質が、該キチン結合ドメインのN末端側およびC末端側からなる群より選択される少なくとも1つの末端側に融合されている、
方法。
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JP2008165781A JP5288902B2 (ja) | 2008-06-25 | 2008-06-25 | キチン結合ドメインを用いる細胞表層提示方法 |
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JP2010004774A JP2010004774A (ja) | 2010-01-14 |
JP5288902B2 true JP5288902B2 (ja) | 2013-09-11 |
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JPS60164477A (ja) * | 1984-02-03 | 1985-08-27 | Agency Of Ind Science & Technol | カビの細胞壁の除去方法 |
US5258502A (en) * | 1989-01-30 | 1993-11-02 | Massachusetts Institute Of Technology | Immobilization and purification of fusion proteins using chitin-binding ability |
JP4796840B2 (ja) * | 2005-12-27 | 2011-10-19 | Bio−energy株式会社 | 糸状菌においてタンパク質を分泌生産する方法 |
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