在啤酒酵母菌內利用重組基因方式尋找CaENO1上的分泌訊號位置

Abstract

真核生物中，大部份的分泌性蛋白質含有一段約10~30個疏水性的胺基酸序列，且通常出現在蛋白質N端；透過辨識這類的訊號，細胞才能將正確的蛋白質分泌至胞外。然而，近年來有相關研究發現，許多真核模式生物的蛋白質不帶有典型N端的分泌訊號胜□(N-terminal signal peptide)，卻可被運輸至細胞表面甚至胞外；而本實驗白色念珠菌(Candida albicans)的烯醇化□(Enolase)即屬此類蛋白質。在白色念珠菌中，烯醇化□主要是由CaENO1基因所表達，大部份存在於細胞質內，為胞內的醣解酵素之ㄧ；有趣的是，在細胞表面及胞外培養基中，也可偵測到此蛋白質的存在。本論文因此利用啤酒酵母菌(Saccharomyces cerevisiae)表達系統，研究烯醇化□是否存在決定性的分泌訊號序列，藉以探討上述現象。首先，必須先確立啤酒酵母菌的系統可以表達外源的念珠菌CaENO1基因，於是將建構好的質體轉形至酵母菌胞內表達，再利用西方點墨(western blot)偵測蛋白質的表現；結果顯示念珠菌CaENO1基因不但成功的在酵母菌內表達，且可被分泌至胞外。確定表達系統建立後，利用建構不同的CaENO1片段並在C端接上螢光蛋白(EGFP)，建構的片段長度以DNA序列表示, CaENO1的1-150 bp, 1-279, 1-387, 1-450,1-510, 1-900, 280-1320, 388-1320, 451-1320, 901-1320, 451-901共11個不同建構產物,並建構一對照組僅有EGFP,比較各個的表現來探討此基因的序列與分泌表現的相關性。透過西方點墨法偵測結果，只有全長的CaENO1 基因所表現之蛋白質可於胞外被偵測；另外，共軛焦顯微鏡下觀察，並未在細胞壁及細胞膜上觀察到enolase-EGFP蛋白質，而是主要位於胞質內。

The evidence of proteins at the yeast cell surface that lack N-terminal signal peptides was initially provided by morphological, biochemical and genetic studies. The existence of many such proteins has subsequently been demonstrated by proteomic approaches. In Candida albicans, the gene encoding enolase is named CaENO1. Enolase is an enzyme of glycolysis and gluconeogenesis as well as major cell-surface antigen, which binds host plasmin and plasminogen. It is immunoprotective, phagocytosis, biofilm-regulated, and farnesol-down regulated. Enolase is detected on the cell surface even in the culture medium, but the mechanism of secretion is still unknown. My study was focused on identifying the critical region of CaENO1 for secretion. First step is to test whether CaENO1 can be expressed in Saccharomyces cerevisiae, CaENO1 was tagged with HA3HIS6 and EGFP, respectively for detecting the target protein. Second is to further analysis the protein secretion. For determining which region is critical, the CaENO1 was truncated for obtaining the constructs with different fragments 1-150 bp, 1-279, 1-387, 1-450, 1-510, 1-900, 280-1320, 388-1320, 451-1320, 901-1320, 451-901 of CaENO1 and negative control EGFP only construct, and then analyzed for the secretion of truncated CaENO1-EGFP protein with western blot. According to the result of western blot, only full length CaENO1 can lead the tagged protein outside the cell. Using confocal laser scanning microscopy, the eno-EGFPp was localized in the cytoplasm but not in the cell membrane or cell wall.the. S. cerevisiae cell seems not recognize the eno-EGFPp as a cell wall protein in this study.