異源蛋白於枯草桿菌宿主之表現研究 : 一、枯草桿菌共同表現之初步探討 ; 二、抗凍蛋白、綠螢光蛋白之組胺酸標幟對其於枯草桿菌表現分泌之影響

Abstract

中文摘要 (第一部分) 枯草桿菌 ( Bacillus subtilis )具有能夠分泌大量的酵素至培養液的特性，可以簡化純化的繁瑣過程，並利於大量酵素生產，因此常被應用於工業上。此外由於Bacillus subtilis具有分泌系統，也常被作為生產外源蛋白的宿主。 外源蛋白於宿主中之表現有時候必須於一宿主中進行多個基因之表現，例如: 為了得到由兩個次單元所構成而具有功能之重組蛋白，或是所欲表現之蛋白進行表現時，必須配合幫助其正確摺疊使其具有活性之因子，此外共同表現轉錄調控相關因子，可研究轉錄機制或提高外源蛋白的產量。 以質體進行基因之共同表現一般有兩種方式，一為使用單一個質體，將所要表現之基因置於同一質體上；另一方法為使用兩個質體進行，將欲表現之基因分別置於兩個不同質體上。本研究中所使用方法是後者，以兩個質體進行共同表現。 本研究中所用的質體主要為pME、pHG及pEX5B。pME是源自於枯草桿菌及大腸桿菌之穿梭載體pMK4，於其上嵌入鹼性彈性蛋白酶subtilisin YaB之結構基因而成；而pHG及pEX5B是於枯草桿菌及大腸桿菌之穿梭載體pHY300PLK上分別嵌入環狀糊精葡萄糖基轉移酶及鹼性彈性蛋白酶subtilisin YaB之結構基因而成。將pME與pEX5B或是pME與pHG同時以電轉方式送入Bacillus subtilis DB104宿主中，以研究pHY300PLK及pMK4為來源的質體，共同表現之可行性。結果顯示以pHY300PLK及pMK4為來源的質體進行共同表現，於兩質體上之基因都可各自表現，且此兩質體之質體穩定度及結構穩定度良好。因此，以pHY300PLK及pMK4為來源的質體於Bacillus subtilis DB104宿主中進行共同表現是可行的。之後可以此兩質體進行其他相關基因之研究。 中文摘要(第二部分) 枯草桿菌最廣為熟知的特性即在其具有很好的胞外分泌系統，可以分泌大量蛋白酶至胞外。然而以枯草桿菌作為分泌表現系統固然有其優點，但往往實際進行外源蛋白於枯草桿菌表現後難以達到預期的結果。 本研究中探討組胺酸標幟對外源蛋白在枯草桿菌的表現及分泌的過程是否會造成影響。本實驗室之前於枯草桿菌中進行抗凍蛋白之分泌表現，將抗凍蛋白與鹼性蛋白酶之訊息融合，鹼性蛋白酶之訊息已證實可於枯草桿菌中有效分泌，但實驗結果難以觀察抗凍蛋白之表現量，之後又更換最佳化之σA型強力啟動子進行表現，實驗結果可測得抗凍蛋白之表現量但發現無法分泌至胞外。由於抗凍蛋白之 C端融合有純化用之組胺酸標幟，帶有多個正電荷，可能對分泌造成影響。本研究以綠色螢光蛋白質及抗凍蛋白為研究對象，研究his-tag對綠螢光蛋白質及抗凍蛋白於Bacillus subtilis中表現及分泌之影響。實驗結果抗凍蛋白於Bacillus subtilis DB430中之表現量難以以SDS-PAGE觀察，而西方轉漬偵測抗凍蛋白結果對於小分子量之抗凍蛋白無法產生抗體免疫反應。而綠螢光蛋白質表現結果存在於胞內成分，且具有his-tag之綠螢光蛋白質表現量較高，之後藉由外加蛋白酶方式對綠螢光蛋白進行穩定度測試，結果發現帶有histidine-tag之綠螢光蛋白對外加之蛋白酶稍有較高之抗性。關於histidine-tag之存在對綠螢光蛋白於B. subtilis DB430中之分泌表現並非影響分泌之主要因素，帶有及不帶有histidine-tag之綠螢光蛋白在鹼性彈性蛋白酶之訊息之協助之下都無法分泌至培養液中。

Abstract (part I) Bacillus subtilis can secrete the enzymes into the supernatant in large amounts and has been used in the production of industrial enzymes for a long time. It is also as excellent host for the secretory production of heterologous proteins. Coexpression of two structure genes in one host is necessary in some cases. One is to get a functional recombinant protein which is consisted of two subunits. Others such as the protein of interest must be coproduced with accessory factors such as molecular chaperones and transcription activation factors to enhanced the yield of active heterologous proteins. There are mainly two strategies to coexpress heterologous proteins. One is to use a bicistronic vector, which is generated by cloning two target genes into one plasmid. The other strategy is to use two plasmids in which foreign genes are cloned in two different vectors. In this study, we use two plasmid coexpression system to ease the control of heterologous protein production. Plasmids used in this study included pME, pHG and pEX5B. The plasmid pME came from the shuttle vector pMK4 with part of subtilisin YaB gene, ale E inserted into it. pEX5B and pHG came from the shuttle vector pHY300PLK, with subtilisin YaB gene and CGTase gene inserted into it separately. pHG or pEX5B was transformed into Bacillus subtilis DB104 with pME. Our goal is to study the possibility of coexpression with pHY300PLK-based vector and pMK4-based vector in Bacillus subtilis. The results suggested that pHY300PLK-based vector and pMK4-based vector can coexpress heterologous proteins in Bacillus subtilis DB104. The segregation and structure stability are high. Based on this coexpression system, other coexpression system can be developed in the future. Abstract ( part II ) Bacillus subtilis can secrete proteins directly into the growth medium, this ability makes Bacillus subtilis an attractive host for expression of heterologous recombinant proteins. Although Bacillus subtilis has this advantage over other host such as Escherichia coli, its secretion of heterologous proteins is not so efficient in many cases. In this study, we aimed to study the effects of histidine-tag to the expression and secretion of heterologous proteins in Bacillus subtilis. In our prior studies, the signal peptide of Subtilisin YaB was fused to the N terminus of the type I antifreeze protein. Although the signal peptide can lead Subtilisin YaB to cell membrane for processing during secretion in Bacillus subtilis. The expression level of antifreeze protein was difficult to observe. Then, we replaced the original promoter with a strong σA type promoter. We found that the expression of the antifreeze protein elevated but located in the cell membrane and insoluble fraction. In this study, we questioned the effect of histidine-tag in the C-terminal regions of antifreeze protein and green fluorescent protein in Bacillus subtilis on their secretion. The results show that the antifreeze protein is difficult to be detected by SDS-PAGE, and it is difficult to detect low molecular weight type I AFP by western blotting. The green fluorescent protein expressed in high amount in the total cell lysate of Bacillus subtilis. And the expression level of the green fluorescent protein with histidine-tag is higher. In vitro protease assay shows that the green fluorescent protein with histidine-tag is more resistant to protease than the GFP without histidine-tag. Histidine-tag is not the main factor to block green fluorescent protein secretiion in Bacillus subtilis DB430.