Development of a Novel Gene Expression System for Secretory Production of Heterologous Proteins via the General Secretory (Sec) Pathway in Corynebacterium Glutamicum
Development of a Novel Gene Expression System for Secretory Production of Heterologous Proteins via the General Secretory (Sec) Pathway in Corynebacterium Glutamicum
复制标题
DOI：
10.21859/ijb.1746
复制
发表时间：
2018-03
影响因子：
1.3
通讯作者：
Huimin Jia;Hedan Li;Lirong Zhang;Daqing Xu
中科院分区：
文献类型：
--
作者：
Huimin Jia;Hedan Li;Lirong Zhang;Daqing Xu
背景谷氨酸棒杆菌（Corynebacterium glutamicum，C.谷氨酸菌）是分泌生产异源蛋白的潜在宿主。然而，迄今为止，很少有分泌型基因表达系统在C。谷氨酸棒杆菌的开发，限制了棒杆菌的应用。谷氨酸杆菌在异源蛋白质的分泌生产中的作用。目的建立一种新的、高效的、依赖于一般分泌途径的异源蛋白表达系统。谷氨酸。材料与方法将合成的克隆表达盒C组装到基本的E.大肠杆菌谷氨酸穿梭载体pAU 2，产生Sec依赖型基因表达载体pAU 5。最后，对C.以枯草芽孢杆菌（Bacillus subtilis）的α-淀粉酶AmyE为报告蛋白，对谷氨酸/pAU 5系统进行了检测。结果成功构建pAU 5载体。SDS-PAGE结果显示，在14067/pAU 5-amyE的原培养上清中可观察到AmyE蛋白条带。Western blotting实验表明，在14067/pAU 5-amyE的培养上清中检测到AmyE多肽，而在14067/pAU 5-amyE的细胞裂解液中检测不到AmyE多肽。14067/pAU 5-amyE细胞培养上清的α-淀粉酶比活力为103.24±7.14 U·mg-1蛋白，而14067/pAU 5-amyE细胞匀浆上清中未检测到α-淀粉酶活性。这些结果表明，重组AmyE在C. glutamicum/pAU 5系统。结论构建了一种新型高效的SEC依赖型基因表达载体pAU 5。谷氨酸。载体pAU 5采用强启动子tac-M控制目的基因的组成型转录，C.谷氨酸梭菌中保证蛋白质翻译的Sec型cgR_2070信号序列和介导蛋白质分泌的Sec型cgR_2070信号序列。谷氨酸。梭谷氨酸/pAU 5系统是一种高效的外源蛋白分泌表达系统。
Background Corynebacterium glutamicum (C. glutamicum) is a potential host for the secretory production of the heterologous proteins. However, to this date few secretion-type gene expression systems in C. glutamicum have been developed, which limit applications of C. glutamicum in a secretory production of the heterologous proteins. Objectives In this study, a novel and efficient general secretory (Sec) pathway-dependent type gene expression system for the production of heterologous proteins was developed in C. glutamicum. Materials and Methods The synthesized cloning/expression cassette C was assembled into the basic E. coli-C. glutamicum shuttle vector pAU2, generating the Sec-dependent type gene expression vector pAU5. Subsequently, the applicability of the C. glutamicum/pAU5 system was tested using the α-amylase AmyE from Bacillus subtilis as a reporter protein. Results The vector pAU5 was successfully constructed. The SDS-PAGE experiment showed the AmyE protein band could be observed in the original culture supernatant of the 14067/pAU5-amyE. The Western blotting experiment showed that the AmyE polypeptide could be detected in the culture supernatant of the 14067/pAU5-amyE, not in the cell lysate of 14067/pAU5-amyE. The α-amylase specific activity of the culture supernatant of 14067/pAU5-amyE was 103.24±7.14 U.mg-1 protein, while no α-amylase activity was detected in the cell homogenate supernatant of 14067/pAU5-amyE. These results demonstrate that the recombinant AmyE was efficiently expressed and completely secreted into the extracellular environmentin an active form in C. glutamicum/pAU5 system. Conclusions A novel efficient Sec-dependent type gene expression vector pAU5 was constructed in the C. glutamicum. The vector pAU5 employs the strong promoter tac-M for controlling a constitutive transcription of the target gene, the consensus ribosome binding site (RBS) sequence of C. glutamicum to ensure protein translation, and the efficient Sec-type cgR_2070 signal sequence to mediate protein secretion in the C. glutamicum. The C. glutamicum/pAU5 system is an efficient expression system for the secretory production of the heterologous proteins.