建立高重組蛋白表達之酵母菌Pichia Pastoris系統

Abstract

利用真核表現系統來製造生產重組蛋白質已被廣泛應用於醫藥、食品與化學工業等方面，嗜甲醇酵母菌Pichia pastoris是目前最常被用來大量表達重組蛋白質的真核表達系統，但由於不同外源基因在P. pastoris中經常會有不同的重組蛋白質表達量，為了要建立一個能穩定且大量表達重組蛋白質的系統，有許多研究報告提出增加外源基因在P. pastoris表達量的方法。本研究採用操縱細胞整體轉錄作用（global transcription machinery engineering）的技術，以提升Candida rugosa重組脂肪酶在P. pastoris的表達量，藉由隨機突變P. pastoris的重要轉錄因子SPT15（一種TATA-binding protein），達到改變P. pastoris的轉錄體（transcriptome），進而影響Candida rugosa重組脂肪酶（lipase）的表達量。利用高通量（high-throughput）脂肪酶活性測定法，挑出具有高脂肪酶表達量的突變株。目前已從464個突變株當中，篩選出16個突變株其脂肪酶表達量高於野生型1.5倍以上，其中最高的一株表達量為野生型的2.3倍。此結果顯示操縱P. pastoris整體轉錄作用可以提升外源重組蛋白在P. pastoris的表達量。

Production of recombinant proteins in eukaryotic system has been widely applied in pharmaceutical, food and chemical industries. Pichia pastoris is one of the mostly used eukaryotic systems to overexpress the recombinant protein. However, not all foreign genes can highly express in P. pastoris system. Many reports tried to establish a reliable high-level expresssion system of P. pastoris. In this study, we used global transcription machinery engineering (gTME) approach to improve the expression level of the Candida rugosa lipase (CRL) in P. pastoris. Random mutagenesis of the transcription factor SPT15 (the P. pastoris TATA-binding protein) was performed to reprogram the transcriptome and to affect the CRL production in P. pastoris. By high-throughput lipase activity assay, 464 mutants were screened to obtain 16 mutants whose lipase productions were improved at least 1.5-fold than the wild type. The highest one displayed 2.3-fold activity than the wild type. It suggests that the expression level of foreign genes could be improved in P. pastoris system through gTME approach.