Abstract
In this study, the potential for heterologous protein expression and secretion via twin-arginine translocation (Tat) pathway was investigated in Escherichia coli DH5α host using enhanced green fluorescent protein (EGFP) as model protein reporter. To construct the shuttle vector pBT2-ET-5X-EGFP, 17 kinds of PCR-amplified 5x-egfp fragments were, respectively, cloned into plasmid pBT2-Peftu-Tat-EGFP and transformed into E. coli DH5α host. By SDS-PAGE, fluorescence microscope observation and flow cytometry analyzation, EGFP was expressed in an active form in the cells of E. coli DH5α, but failed to translocate to the culture medium.
B. Xu and Y. Cheng—Co-first authors.
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Acknowledgments
We are very grateful to Prof. Dr. Friedrich Goetz of University of Tuebingen, Germany, for kindly providing plasmid pBT2, we also thank Ms. Lin Huang and Mr. Hao Zhou for excellent technical assistance and instrument support. This work was sponsored by the Natural Science Foundation of China (31370075 & 31471725), the National 973 Program of China (2013CB734004), and the National 863 Program of China (2012AA021302).
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Xu, By. et al. (2015). Construction of Eschericha coli-Staphylococcus Shuttle Vector for EGFP Expression and Potential Secretion via Tat Pathway. In: Zhang, TC., Nakajima, M. (eds) Advances in Applied Biotechnology. Lecture Notes in Electrical Engineering, vol 333. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-662-46318-5_19
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DOI: https://doi.org/10.1007/978-3-662-46318-5_19
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