最佳σA啟動子及多重啟動子之構築及其於枯草桿菌中的表現

Abstract

中文摘要 本研究利用不同種類的啟動子，構築一系列含有強力 σA 啟動子及多套式啟動子(σA + σB )之表現載體，並配合枯草桿菌之菌體外分泌系統，以期提高本實驗室所合成之第一型抗凍蛋白（typeⅠantifreeze proteins）以及鹼性彈性蛋白(alkaline elastase)之表現量。 以重疊延展聚合鏈鎖反應(OEPCR)合成最佳之枯草桿菌σA啟動子，並與枯草桿菌進入產孢期後之壓力反應σB啟動子，比較其重組蛋白之表現量。同時考慮枯草桿菌之菌體外分泌系統能力，製備成多重啟動子(complex promoter)，該啟動子包含σA及σB兩組啟動子。由蛋白基因及第一型抗凍蛋白基因表現量發現，σA啟動子可於不影響菌體正常生長之情況下，提高重組蛋白之表現，且由5’ RACE及mRNA二級結構之分析結果亦顯示σA啟動子之轉錄及轉譯效能較佳，可提早表現蛋白質，並藉由mRNA穩定之二級結構更有助於σA啟動子大量表現重組蛋白質。此外，σB啟動子則在較晚期表現，且表現量較低。而多重啟動子之組合並未如預期使目標基因可持續強力的表現，其表現量反而低於σA及σB啟動子。根據RACE之結果推測與啟動子之使用頻率有關，整個重組蛋白之表現過程中，大多為σB啟動子在驅動目標基因之表現，故其蛋白質表現量較單獨使用σA啟動子之表現量低。另一方面，抗凍蛋白僅存於枯草桿菌菌體內並未如期分泌至胞外培養基中，且其主要存在於細胞膜成分及不可溶部份。推測可能為分泌機制所造成之影響所致。本實驗室所構築之σA啟動子表現系統確實可有效提高重組蛋白質之表現、分泌量。未來可利用此系統來生產有價值之異源性蛋白質以供研究、應用之所需。

Abstract Bacillus subtilis is one of the most efficient expression hosts for recombinant proteins. They are well known for the ability to secrete a wide variety of extracellular proteins. The efficient secretion system can be used to produce commercially important heterologous proteins economically superior to the commonly used host Escherichia coli. To improve the productivity of B. subtilis, efforts have been put mainly on two aspects: development of protease-deficient strains and application of efficient regulatory elements at both transcription and protein secretion levels. Here, we synthesize a strongσA —type promoter for excellent expression of proteins in B. subtilis, and a complex σA — andσB -type promoter to optimize the extracelluar yields of antifreeze proteins and alkaline elastase YaB in B. subtilis DB104. The promoters are designed according to the collection and analysis of many reports and data bases. Using overlapping extension polymerase chain reaction (OEPCR) method to synthesize the designed σA —type promoter and σA + σB complex promoter. These DNA fragments are inserted into pEX5B、pAFPB and pRPAFPB to construct a series of expression vectors. The experimental data show that the σA — type promoter can improve the yields of recombinant proteins without influencing the normal growth of B. subtilis DB104. Furthermore , the results of 5’ RACE and mRNA secondary structure prediction also reveal that excellent transcription efficiency and stable mRNA secondary structure of σA — type promoter increase the proteins expression yields. In addition , the lower expression yields of σA + σB complex promoter could be the σB — type promoter utilizing frequency is higher than σA — type promoter during the period of protein expression. AFPs mainly exist in total cell lysate of B. subtilis DB104, especially in cell membrane and insoluble fraction. The reason of unsuccessful extracellular secretion could be due to the limit of secretion machinery. However, the σA —— type promoter expression system increase the expression yields of recombinant proteins and considered as a potent system for other recombinant protein expression.