增進第一型重組抗凍蛋白質類似物於乳酸鏈球菌之分泌表現

Abstract

近年來乳酸菌的相關研究備受矚目，相關文獻的發表更是一日千里，其中不乏以基因工程方式改造乳酸菌的應用。Lactococcus lactis 為目前研究最廣泛之乳酸菌菌株，因其屬GRAS（Generally recognized as safe）級安全菌株，故以其作為異源蛋白質之表現宿主具有應用於食品工業上之潛力。本論文中即以L. lactis NZ9000 作為宿主，重組抗凍蛋白類似物作為模式蛋白質，期望建立一高產量之食品級分泌表現系統。抗凍蛋白質（antifreeze proteins，AFPs）也稱作熱遲滯蛋白（thermal hysteresis proetins，THPs），存在於某些寒帶或極地生物體內，具有降低凍結點及抑制冰晶再結晶的特性，可應用於冷凍食品之品質保持或細胞、組織之冷凍保存上。本實驗室先前已利用人工合成的方式，成功將來自於魚類之第一型重組抗凍蛋白質類似物表現Escherichia coli、Bacillus subtilis 及L. lactis 中。本論文將進一步探討重組抗凍蛋白質類似物在L. lactis 中之分泌表現量，並將表現元件置換為GRAS 級來源以朝食品級載體構築之方向邁進，最後開發一成份安全之培養基（FMB）以作為食品級表現之用。論文中首先探討來自於L. lactis secreted protein（Usp45）、B. subtilis levansucrase（SacB）、B. subtilis YaB alkaline elastase subtilisin（Ale）及Lactobacillus acidophilus S-layer protein（SlpA）四種蛋白質之訊息胜肽對重組抗凍蛋白質類似物分泌表現量之影響，結果顯示SacB之訊息胜肽在L. lactis NZ9000 中對重組抗凍蛋白質有最佳之分泌量，且由實驗結果發現若在訊息胜肽C端添加Ala及Glu兩胺基酸可提升重組抗凍蛋白質之分泌量。故後續實驗選用SacB之訊息胜肽，並比較在訊息胜肽C端添加Ala 、Glu （ AE ） 或先前已有文獻報導之分泌促進胜肽Leu-Glu-Ile-Ser-Ser-Thr-Cys- Asp-Ala（LEISSTCDA），對蛋白質分泌量之影響。結果顯示在SacB訊息胜肽C端添加AE確實可提升重組抗凍蛋白質之分泌量；而添加LEISSTCDA之效果反而不如預期，推測原因可能為重組抗凍蛋白質N端本身即帶有兩個負電，故LEISSTCDA無法發揮其功效。最後選擇在C端添加AE之SacB訊息胜肽作為分泌訊號，並將質體上之轉錄終止子置換成同為GRAS級來源之slpA轉錄終止子（TerslpA），但由於構築過程中屢屢發生突變，無法挑選到序列完全正確之TerslpA，故最後選用了三種在不同鹼基對產生突變之TerslpA加以比較，結果顯示在第24 個位置發生突變之轉錄終止子對重組抗凍蛋白質有最佳之分泌表現量。本實驗成功挑選到一分泌效果較目前常用於L. lactis 之分泌訊號SPusp45-LEISSTCDA佳之SPsacB-AE，且用於表現具活性之重組抗凍蛋白質類似物；開發一成分安全之生物安全級培養基，成功使用於L. lactis大量表現重組抗凍蛋白質類似物。

The researches of the lactic acid bacteria (LAB) receive much concern in recent years. Among the published literatures, the genetically modified LAB and its application are attractive. Lactococcus lactis, a generally recognized as safe (GRAS) bacterium, is the most extensively studied LAB. It is widely used for industrial fermentation and has recently become an attractive host for the production of heterologus proteins. In this study, we used L. lactis NZ9000 as host, aimed to set up a food grade expression-secretion system to produce recombinant antifreeze protein (AFP) analogue. AFPs, also known as thermal hysteresis proteins (THPs), were found in a wide range of organisms living in cold ambient conditions. The ability of AFPs to influence ice crystal growth has led these proteins used potentiality in a wide variety of applications, such as use of them as food additives to enhance the quality and shelf-life of frozen foods. In previous studies, a synthetic gene encoding the recombinant type I AFP (rAFP) analogue has been expressed in Escherichia coli, Bacillus subtilis and L. lactis in our labortory. In this study, we have investigated the influence of various signal peptides, transcription terminators, and media on the secretory production of rAFP in L. lactis. In the first section, we examined the effect of the signal peptides from L. lactis secreted protein (Usp45), B. subtilis levansucrase (SacB), B. subtilis YaB alkaline elastase subtilisin (Ale) and Lactobacillus acidophilus S-layer protein (SlpA) in secretion yield of rAFP. Results showed that SPsacB exhibited the best secretion level in L. lactis NZ9000. When AE was fused immediately after the signal peptide cleavage site, secretion yield of rAFP was increased more than fused to LEISSTCDA. By the results, we choose SPsacB and fused AE after cleavage site as the secretion signal to produce rAFP. And then, we replaced the pQE terminator with slpA terminator to achieve the food grade production system. However, the entire accurate TerslpA was unable to obtain and the point mutation in TerslpA were happened during the construction. Therefore, three TerslpA which mutated in different base pair were subject to compare. Result showed that mutated in the 24th position of TerslpA exhibited the best secretion yield in expression of rAFP. A biosafety culture medium was developed to express the rAFP with best secretion and terminator signal at high level in L. lactis.