Team:HokkaidoU Japan/Circularization
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Team:HokkaidoU Japan
Although enzymes are essential for the development of science, they can only be used under certain conditions due to its nature that it inactivates depending on the temperature and pH. Ways to gain enzymes that are stable against such conditions include investigation of microorganisms that survive under extreme environments, improvement of the activation using amino acid substitution, compartmentalization of the enzymes, and circularization of polypeptides. When enzymes are heated or the pH changed, linear-type enzymes are denatured and deactivated. On the other hand, with circularized enzymes, it is believed that since the ends of the polypeptide are joined and protected, the tertiary structure is less likely to be broken and the activation kept. This year, we attempted in the circularization of proteins using self-assembling peptide(SAP) and zip-up linker.
The SAPs we used are RADA-16-I and P11-4. These are both artificially created amphiphilic SAPs, consisting of amino acid sequence RADARADARADARADA and QQRFEWEFEQQ respectively. They self-assemble under suitable physiochemical conditions due to the polar amino acids and hydrophobic interaction and form β-sheet (Fig.1).
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Fig.1 RADA16-I and P11-4 self-assemble under suitable physiochemical conditions due to the polar amino acids and hydrophobic interaction and form β-sheet.
By creating a construction as shown in Fig.2, the self-assembling region(SAR) and the region containing the SAP interact, thus bringing closer the zip-up linkers on the N-terminal and the C-terminal (Fig.3).
Fig.2 Genetic construct used for the circularization of proteins using self-assembling peptide(SAP) and zip-up linker
Fig.3
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The zip-up linker plays a vital role in the creation of the covalent bond essential for the circularization of proteins. This consists of amino acid sequence of CWEGGGCGGGCGGGCSALCGGGCGGGCGGG, and is composed of repetition of 3 glycine and 1 cysteine residues. We are hoping that the zip-up linker on the N and C terminal are brought closer by the SAR, and that the cysteine residues form disulfide bonds from the SAR as if to zip up the ends.
Since the distance between the N-terminal and the C-terminal varies depending on the protein, it is essential that a linker of an appropriate length is chosen. This would usually mean that it is necessary to change the linker depending on the protein that is to be circularized. However, our zip-up linker has enough GGGC sequence so that only the flexible part of the linker form disulfide bonds, thus preventing deformation of the tertiary structure. This means that a suitable length of the linker will be used to suit the structure of each protein, enabling this zip-up linker to be applied to various proteins regardless of its structure. Eventually, a structure as indicated in Fig. 4 is obtained.
今回我々はGFPを用いて環状化したタンパク質の熱およびｐHに対する安定性を評価した。まず、Fig.2における酵素をGFPとしたコンストラクト、およびzip-up linkerやSARの影響を調べるための7種類のネガティブコントロール用コンストラクトを設計した。7つのネガティブコントロール用コンストラクトはそれぞれGFPの上流または下流いずれか一方のSARを除いたもの（a）(b)、GFPの上流または下流いずれか一方のzip-up linkerを除いたもの(c)(d)、上流と下流にzip-up linkerまたはSARのみをつけたもの(e)(f)、およびGFPのみのもの(g)であり、いずれも適当な制限酵素を用いることでFig.1に示したようなGFPと両端のzip-up linker,およびSRAからなる完全なパーツから切り出して作ることが可能である。なお、すべてのコンストラクトには精製のため末尾に6Xヒスチジンタグがつけられている。設計したFig.1のコンストラクトはIDTに合成を発注し、ベクターpSB1C3にのせて大腸菌に導入した。ネガティブコントロールについてはその後さらに適当な酵素でダイジェッションを行って完全体から各部分を適宜抜き出して作成した。次に、コンストラクトをのせたベクターを導入した大腸菌に対してIPTG誘導を行い、各パーツを発現させたのち、凍結融解により大腸菌を溶菌させて遠心分離を行い、得られたタンパク質分画からNi アフィニティーカラムクロマトグラフィーを利用して目的のタンパク質を精製した。さらに、精製したタンパク質を環状化させる工程として（SARの自己組織化する培養条件かく）でインキュベートし、SARの自己組織化を促進したのちGST（略さない名称、機能）で処理してzip-up linker間のジスルフィド結合形成を促進した。（以下ヒートショック方法、安定性評価法かく）
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