基于Split-GFP系统定量分析大肠杆菌中异源表达的类胶原蛋白Scl2  

Quantitative Analysis of Collagen-Like Protein Scl2 Heterologously Expressed in Escherichia coli Based on Split-GFP System

作　　者：色依德·斯马依 赵晨旭 张轶群 刘业学 王稳航[2] 李玉[1] SIMAYI Seyide;ZHAO Chenxu;ZHANG Yiqun;LIU Yexue;WANG Wenwang;LI Yu(Key Laboratory of Industrial Fermentation Microbiology,Ministry of Education,College of Biotechnology,Tianjin University of Science and Technology,Tianjin 300457,China;College of Food Science and Engineering,Tianjin University of Science and Technology,Tianjin 300457,China)

机构地区：[1]工业发酵微生物教育部重点实验室,天津科技大学生物工程学院,天津300457 [2]天津科技大学食品科学与工程学院,天津300457

出　　处：《天津科技大学学报》2025年第1期13-19,27,共8页Journal of Tianjin University of Science & Technology

基　　金：国家重点研发计划项目(2023YFF1103901)。

摘　　要：利用大肠杆菌(Escherichia coli)表达类胶原蛋白Scl2,并通过Split-GFP系统建立一种简便、快速且可定量检测Scl2的方法。结果表明,Scl2在大肠杆菌(Escherichia coli)BL21(DE3)中成功表达,并通过His亲和标签纯化得到较高纯度的Scl2。圆二色光谱和差示扫描量热仪分析发现,Scl2的二级结构和热稳定性与动物源Ⅰ型胶原蛋白相似,并且GFP11的融合对其几乎没有影响。GFP1-10和Scl2-GFP11结合的前20 h的结合速度较高,达到最大相对荧光强度需要约70 h。当两者结合1 h时,Scl2-GFP11蛋白质量浓度与相对荧光强度之间能够形成较好的线性关系,相关系数R2为0.9995,重复性良好。本研究利用Split-GFP系统建立了体外检测并定量分析Scl2的方法,为下一步高通量筛选研究提供了快速、简便的工具。In the current study,Scl2 was expressed in Escherichia coli BL21(DE3),and a simple and efficient detection method was also established for quantitative analysis of Scl2 through the Split-GFP system.The results indicated that Scl2 was successfully expressed in E.coli BL21(DE3)and purified with the use of His affinity label,resulting in high purity Scl2.CD spectroscopy and DSC analysis demonstrated that the secondary structure and thermal stability of Scl2 were comparable to those of animal-derived typeⅠcollagen.The presence of GFP11 had minimal impact on these properties.The first 20 hours of binding between GFP1-10 and Scl2-GFP11 showed a high binding rate and it took about 70 hours to reach the maximum fluorescence intensity.After 1 hour of binding,a positive linear relationship was observed between the concentration of Scl2-GFP11 protein and fluorescence intensity.The correlation coefficient R2 was calculated to be 0.9995,indicating excellent repeatability.Therefore,we successfully established a method for in vitro detection and quantitative analysis of Scl2 using the Split-GFP system,which has provided a fast and convenient tool for future high-throughput screening research.

关 键 词：大肠杆菌 异源表达 类胶原蛋白Scl2 Split-GFP 蛋白定量分析 

分 类 号：Q816[生物学—生物工程]