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Secreted production of Renilla luciferase in Bacillus subtilis
Author(s) -
Chiang ChungJen,
Chen Po Ting,
Chao YunPeng
Publication year - 2009
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1002/btpr.351
Subject(s) - bacillus subtilis , heterologous , secretion , luciferase , plasmid , chemistry , secretory protein , gene , transcription (linguistics) , reporter gene , promoter , signal peptide , mutant , microbiology and biotechnology , gene expression , biochemistry , biology , transfection , bacteria , recombinant dna , genetics , linguistics , philosophy
Luciferase (Rluc) from the soft coral Renilla reniformis has been widely used as a bioluminescent reporter, and its secreted production has been solely performed in mammalian cells thus far. To make the production more efficient, a series of approaches was attempted to overproduce Rluc extracellularly in Bacillus subtilis . First, Cys124 in the Rluc gene was substituted with Ala. The mutant gene was subsequently incorporated into a pUB110/R6K‐based plasmid, consequently, fusing with the P43 promoter and the sacB signal peptide. With the nitrogen‐rich medium, B. subtilis strain bearing the plasmid became able to secret a detectable amount of Rluc. Moreover, the secretion signal for the Rluc gene was replaced by the aprN leader peptide with or without the propeptide. The result led to a more than twofold increase in the secreted Rluc. Finally, by enhancing the transcription of the Rluc gene implementing the P43 and spac tandem promoter, it resulted in the secreted Rluc with a yield of 100 mg/L. Overall, this study illustrates a potential strategy for improving the secretion efficiency of heterologous proteins in B. subtilis . © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010