Protein engineering for conferring a biological function on ovalbumin

赋予卵清蛋白生物学功能的蛋白质工程

• 批准号: 15380229
• 负责人: HIROSE Masaaki
• 金额: $ 7.68万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15380229/
• 关键词: ovalbumin; serpin; loop insertion; conformation change; protease inhibition; タンパク質工学; 卵白タンパク質; セリンプロティナーゼ

Ovalbumin is a member of serpin superfamily, but it does not have any proteinase inhibitor activity. We have done site-directed mutagenesis approach to confer serpin function on ovalbumin. We found by X-ray crystallography that an ovalbumin mutant R339T has an ability to undergo a serpin loop insertion after the P1-P1' cleavage by elastase. This was an important finding for the achievement of our aim, but the mutant did still not have an inhibitory activity against a protease. For further mutagenesis approach, we created a different ovalbumin mutant R339T/A352R in which the P1-P1' site is accessible against trypsin. Utilizing the mutant, a reliable HPLC analysis for the determination of the loop insertion rate was established. Because of the structural and functional situations of serpin, increased loop insertion rate should lead to the acquisition of the inhibitory activity. We therefore did further mutagenesis to accelerate the loop insertion rate on the basis of the data of crystal structure. Alternative mutants, K290T/A352R/R339T and R104/A352R/R339t, and the disulfide-reduced form of A352R/R339T, respectively, displayed 1.5, 3.7, and 6.7- fold increase in the loop insertion rate as compared A352R/R339T control. The mutation and disulfide reduction should give a more flexible nature on the distal sheet A structure. We therefore concluded that the transition state of the serpin loop insertion reaction assumes an opened sheet A conformation.

卵清蛋白是丝氨酸蛋白酶抑制剂超家族的成员，但它不具有任何蛋白酶抑制剂活性。我们采用定点诱变方法赋予卵清蛋白丝氨酸蛋白酶抑制剂功能。我们通过 X 射线晶体学发现，卵清蛋白突变体 R339T 能够在弹性蛋白酶 P1-P1' 裂解后进行丝氨酸蛋白酶抑制剂环插入。这是实现我们目标的重要发现，但突变体仍然不具有针对蛋白酶的抑制活性。为了进一步诱变方法，我们创建了不同的卵清蛋白突变体 R339T/A352R，其中 P1-P1' 位点可抵抗胰蛋白酶。利用突变体，建立了可靠的 HPLC 分析来确定环插入率。由于丝氨酸蛋白酶抑制剂的结构和功能情况，增加的环插入率应该导致获得抑制活性。因此，我们在晶体结构数据的基础上进行了进一步的诱变以加快环插入速率。与 A352R/R339T 对照相比，替代突变体 K290T/A352R/R339T 和 R104/A352R/R339t 以及二硫键还原形式的 A352R/R339T 环插入率分别增加了 1.5、3.7 和 6.7 倍。突变和二硫键还原应赋予远端片A结构更灵活的性质。因此，我们得出结论，丝氨酸蛋白酶抑制剂环插入反应的过渡态呈现打开的A片构象。

项目成果

Dynamic mechanism for the serpin loop insertion as revealed by quantitative kinetics.

定量动力学揭示了丝氨酸蛋白酶抑制剂环插入的动态机制。

• 发表时间: 2005
• 期刊: Journal of Molecular Biology 348・2
• 作者: A.Shimada;H.Yamane;Y.Kimura;Tomomi Imamura;Tomomi Imamura;N.Takahashi;N.Takahashi
• 通讯作者: N.Takahashi

Egg white proteins-ovalbumin. in Progress in Biotechnology 23 (Industrial Proteins in Perspective)(ed. Aalbersberg et al.)

蛋清蛋白质——卵清蛋白。

• 发表时间: 2003
• 作者: Hamasu;T.;Inanami;O.;Tsujitani;M.;Yokoyama;K.;Takahashi;E.;Kashiwakura;I.;Kuwabara;M.;A.Kondo et al.;井原正隆他2名;M.Shimada;M.Hirose
• 通讯作者: M.Hirose

Progress in Biotechnology 23(Indus-trial Proteins in Perspective, ed. Aalversberg et al.)

生物技术进展 23（工业蛋白质展望，Aalversberg 等编辑）

• 作者: Endo;Y;et al.;井原正隆他3名;M.Hirose
• 通讯作者: M.Hirose

Masayuki Yamasaki: "Crystal structure of S-ovalbumin as a Non-loop-inserted Thermostabilized Serpin Form"The Journal of Biological Chemistry. 278・37. 35524-35530 (2003)

Masayuki Yamasaki：“非环插入热稳定丝氨酸蛋白酶抑制剂形式的 S-卵清蛋白的晶体结构”生物化学杂志 278・37（2003）。

Crystal Structure of S-ovalbumin as a Non-loop-inserted Thermostabilized Serpin Form*

• DOI: 10.1074/jbc.m305926200
• 发表时间: 2003-09
• 期刊: Journal of Biological Chemistry
• 影响因子: 4.8
• 作者: M. Yamasaki;N. Takahashi;M. Hirose