CAJ | 학술논문

目的:探讨胰高血糖素样肽-1(GLP-1)的序列拷贝数及拷贝温度、时间与其体外表达水平的关系.方法:首先将以4拷贝数为单位的GLP-1序列插入到pET-22b(+)中构建GLP-1多拷贝表达载体,然后将表达载体转化至大肠杆菌BL21(DE3)中进行蛋白体外培养表达.取不同温度和诱导时间段进行蛋白表达,检测GLP-1在不同条件下的蛋白表达量,优化蛋白最适表达条件. 结果:GLP-1多拷贝在BL21菌株中的最佳表达温度为26℃,最适诱导时间为8 h. 拷贝数与GLP多拷贝表达量呈负相关性,拷贝数越多,蛋白产量越低. 结论:GLP多拷贝最佳体外表达条件为4拷贝,26℃培养IPTG诱导8 h.
목적:탐토이고혈당소양태-1(GLP-1)적서렬고패수급고패온도、시간여기체외표체수평적관계.방법:수선장이4고패수위단위적GLP-1서렬삽입도pET-22b(+)중구건GLP-1다고패표체재체,연후장표체재체전화지대장간균BL21(DE3)중진행단백체외배양표체.취불동온도화유도시간단진행단백표체,검측GLP-1재불동조건하적단백표체량,우화단백최괄표체조건. 결과:GLP-1다고패재BL21균주중적최가표체온도위26℃,최괄유도시간위8 h. 고패수여GLP다고패표체량정부상관성,고패수월다,단백산량월저. 결론:GLP다고패최가체외표체조건위4고패,26℃배양IPTG유도8 h.
Objective:To investigate the relationship between production and copy number coding sequence of the glucagon-like peptide-1 (GLP-1). Methods:Firstly, four repeated sequences for multi-copy GLP-1 peptides were linked as a motif and inserted into the vector pET-22b (+). The completed plasmid including different numbers of GLP-1 copy was subsequently transformed into E.coli BL21 cells, which can be induced to express the interesting peptides by the reagent IPTG (Isopropyl-beta-D-thiogalactopyranoside). The expression of multi-copy GLP-1 was employed to optimize the fermentation. The conditions such as temperature and inducing time for highest protein yield were analyzed. Finally, the effects of different GLP-1 motif were confirmed to improve the production. Results:Multi-copy GLP-1 was expressed and secreted by E.coli in different conditions, the result demonstrated that a climax yield for protein was obtained at 26℃and 8 hours. Moreover, the production was negatively correlated with copy number of GLP-1. With the multi-copy, the production of peptides marked a significant decline. Conclusion:The final optimized condition to express multi-copy GLP-1 peptides is E.coli BL21 containing 4 copy motif vector cultured at 26℃with inducing time of 8 hours.