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Iranian Journal of Biotechnology، جلد ۱۶، شماره ۱، صفحات ۴۱-۴۷
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عنوان انگلیسی Development of A Novel Gene Expression System for Secretory Production of Heterologous Proteins via the General Secretory (Sec) Pathway in Corynebacterium glutamicum
چکیده انگلیسی مقاله 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 effi cient 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 specifi c 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 effi ciently expressed and completely secreted into the extracellular environmentin an active form in C. glutamicum/pAU5 system.Conclusions: A novel effi cient 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 effi cient Sectype 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.
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نویسندگان مقاله Huimin Jia |
College of Life Sciences, Agricultural University of Hebei, Baoding 071001, China

Hedan Li |
College of Life Sciences, Agricultural University of Hebei, Baoding 071001, China

Lirong Zhang |
Biological Control Centre of Plant Diseases and Pests of Hebei, Agricultural University of Hebei, Baoding 071001, China

Daqing Xu |
College of Life Sciences, Agricultural University of Hebei, Baoding 071001, China

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