Cloning, Functional Expression and Characterization of a Phytocystatin Gene from Jelly Fig (Ficus Awkeotsang Makino)Achenes

愛玉瘦果植物性硫氫蛋白酶抑制蛋白基因的選殖、功能性表達與重組蛋白質特性分析

徐志宏(Douglas J. H. Shy) ； 楊翊梅(Yi-Mei Yonug) ； 盧錫祺(Hsi-Chi Lu) ； 鄭月媚(Yueh-Mei Cheng) ； 曾志正(Jason T. C. Tzen) ； 周文敏(Wing-Ming Chou)

《Botanical Studies》 52卷4期 (2011/10) Pp. 407-416

摘要

由成熟愛玉瘦果的cDNA 資料庫中分析約300 個表現序列標籤(EST clone)。其中一cDNA 選殖株之DNA 序列經比對及演譯分析結果，預測為植物性硫氫蛋白酶抑制蛋白(phytocystatin)。此選殖株被命名為FaCYS，全長含有582 bp，可轉譯得到114 個胺基酸。經預測帶有訊息胜肽，成熟的FaCYS 不含任何cysteine，分子量為10.8 kDa，pI 值為9.7。將FaCYS 分別選殖至非融合與融合(his-tagged) 的表達載體中，將重組質體轉殖於大腸桿菌並表達得到可溶性的非融合與融合FaCYS 重組蛋白質。純化後所得之非融合與his-tagged 融合FaCYS 重組蛋白皆具有papain 之抑制活性，其Ki 值極為接近，分別為2.7×10-7 M 與2.4×10-7 M。進一步分析發現his-tagged FaCYS 對人類cathepsin B、cathepsin L 及ficin亦具有抑制活性，其Ki 值分別為5.6×10-7 M、3.0 x10-8 M 和2.0×10-7 M；但對bromelain 則無抑制活性。his-tagged FaCYS 在不同的pH 環境中具有很好的耐受性，亦具有不錯的熱穩定性，於50°C 下30分鐘後仍維持對papain 的抑制活性。此外，此重組蛋白可抑制Glomerella cingulata 及Sclerotium rofsii菌絲的生長。

關鍵字

硫氫蛋白酶抑制蛋白 ； 硫氫蛋白酶 ； 愛玉瘦果 ； 植物性硫氫蛋白酶抑制蛋白 ； 重組蛋白

並列摘要

A cDNA clone encoding a phytocystatin was isolated and identified from about 300 expressed sequence tag (EST) clones in maturing jelly fig (Ficus awkeotsang Makino) achenes. This clone, named FaCYS, consists of 582 bp encoding 114 amino acids with a putative signal peptide. The predicted matureprotein contains no cysteine and has a molecular mass of 10.8 kDa with an isoelectric point (pI) of 9.7. Fa-CYS constructed in nonfusion and fusion vectors were overexpressed in Escherichia coli as nonfusion and histagged recombinants, respectively. Both recombinants were found in the soluble fractions of the cell extracts.The purified nonfusion and his-tagged FaCYS exhibited papain inhibitory activity with similar Ki values of 2.7 × 10-7 M and 2.4 × 10-7 M, respectively. In addition, his-tagged recombinant proteins showed inhibitory activity toward human cathepsin B, cathepsin L and ficin with Ki values of 5.6 × 10-7 M, 3.0 × 10-8 M and 2.0 × 10-7 M, but no inhibitory activity against stem bromelain. It was tolerant at a wide range of pH values and thermally stable up to 50°C for 30 min. Furthermore, his-tagged FaCYS could arrest the fungal growth of Glomerella cingulata and Sclerotium rofsii.

並列關鍵字

Cystatin ； Cysteine protease inhibitor ； Jelly fig achene ； Phytocystatin