臺灣八角蓮方向蛋白在Pichia pastoris之異源表現

Abstract

鬼臼素 (podophyllotoxin) 乃一重要抗癌藥物之前驅物，其生合成起始於松柏醇自由基 (coniferyl alcohol radical) 之雙聚化 (dimerization) 反應而形成松脂醇 (pinoresinol)。方向蛋白 (dirigent protein) 經報導可為此雙聚化反應提供立體結構限制使松柏醇自由基具高度選擇性地形成松脂醇。前人研究中已由臺灣八角蓮 (Podophyllum pleianthum Hance) 中選殖出方向蛋白 cDNA 序列，本研究進一步經由多基因序列比對發現許多功能性相關之保守性胺基酸亦存在於臺灣八角蓮之方向蛋白中，並藉由基因轉形至菸草毛狀根及嗜甲醇酵母菌以建立異源表現平台。其中，嗜甲醇酵母菌轉形株於甲醇誘導後以變性電泳分析培養上清液可得到一特異性表現之蛋白質色帶，未來將經由生物反應器大量培養以收取蛋白質樣品進行後續活性分析。

(-)-Podophyllotoxin is an important lead compound of anti-cancer drugs. Its biosynthesis is initiated with dimerization of coniferyl alcohol radicals to form (+)-pinoresinol. Dirigent protein has been reported structural restriction to provide region- and stereo-selectivity to during the dimerization reaction. In previous study, a cDNA sequence of dirigent protein was cloned from Podophyllum pleianthum Hance. Through multiple sequence alignment, we found that the conserved amino acid residues related to function also exist in the dirigent protein sequence from P. pleianthum Hance. We also tried to construct heterologous expression platforms of dirigent protein in tobacco (Nicotiana tabacum) hairy roots and Pichia pastoris. In the medium supernatant of P. pastoris transformants induced by methanol, a differentially expressed protein band was seen in SDS-polyacrylamide gel electrophoresis. We will further collect protein samples from large-scale fermentation in bioreactors to conduct activity assay.