提高酵母菌 Pichia pastoris 蛋白表達系統的重組蛋白產量

Improvement in the Production of Recombinant Proteins in Pichia pastoris

翁啟翔(Chi-Hsiang Weng)

指導教授 ： 李冠群

國立臺灣師範大學/理學院/生命科學研究所/碩士(2010年)

摘要

酵母菌 Pichia pastoris 已被廣泛應用於外源基因的蛋白表達。此系統具有將重組蛋白分泌至細胞外的特色，又 P. pastoris 本身產生之分泌性蛋白數量少，因而可以藉由簡單的方式，直接純化培養液中的重組蛋白。然而，並非所有重組蛋白本身含有之分泌性序列皆可於 P. pastoris 系統中具有功能，目前發展出許多不同之分泌性序列用以成功將重組蛋白分泌至 P. pastoris 細胞外。故取得高產量之分泌性重組蛋白，首先則必須找尋一最合適之分泌性序列。本研究係藉由篩選九種不同的分泌性訊號序列，找尋一具有高效率的訊號序列，來提高分泌至細胞外的重組蛋白以增加產量，並以 Candida rugosa lipase 作為表達之重組蛋白，利用 lipase 活性測試來篩選出最佳之訊號序列。研究結果發現，該重組蛋白最合適之分泌性序列為來自 Gallus gallus lysozyme 之分泌性訊號序列。此結果顯示，lysozyme 之分泌性訊號序列有助於提升 C. rugosa lipase 在 P. pastoris 表達產量，其活性分析結果為 37 unit / ml，與表現量最差者相差 10 倍。同時，此篩選系統亦可應用於其他重組蛋白於 P. pastoris 系統中的表達，以提升重組蛋白之產量。

關鍵字

Pichia pastoris ； recombinant proteins ； secretion signal sequence

並列摘要

Pichia pastoris is a well known expression system of recombinant proteins, and has been widely used to express various heterologous proteins. It offers the potential of secretion of recombinant protein and simplifying the purification steps because P. pastoris secretes only low levels of endogenous proteins. However, the native secretion signal sequence of a foreign may or may not function properly in P. pastoris. Although several different secretion signal sequences have been used successfully, a choice of the specific secretion signal for a particular target protein must be made to obtain high secretion levels in P. pastoris. In this study, nine secretion signal sequences were respectively incorporated into a P. pastoris vectors which express the recombinant secretory Candida rugosa lipases with different secretion signal peptide. Among the transformants that have single copy of integrated foreign gene, the secretion signal sequence from Gallus gallus lysozyme is suitable for optimal expression and secretion of the recombinant lipase. The protein secretion level of this signal was 10 times higher than the minimal one in this study. The result indicates that secretion signal engineering is feasible to improve the production of a recombinant protein in P. pastoris.

參考文獻

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