請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/62053
標題: Construction of Chromosomally Located T7 Expression System for Production of Heterologous Secreted Proteins in Bacillus subtilis
作者: Chen, P.T.
蕭介夫
Shaw, J.F.
Chao, Y.P.
Ho, T.H.D.
Yu, S.M.
關鍵字: Bacillus subtilis
T7
FLP recombinase
integration
bacteriophage-t7 rna-polymerase
escherichia-coli
recombinant
nattokinase
gene-expression
integration
promoter
dna
replication
bacteria
natto
期刊/報告no:: Journal of Agricultural and Food Chemistry, Volume 58, Issue 9, Page(s) 5392-5399.
摘要: Bacillus subtilis is most commonly employed for secretion of recombinant proteins. To circumvent the problems caused by using plasmids, the T7 expression system known for its high efficiency was rebuilt in B. subtilis. Accordingly, a markerless and replicon-free method was developed for genomic insertion of DNAs. By the act of homologous recombination via the guide DNA, a suicidal vector carrying the gene of interest was integrated into genomic loci of bacteria. Removal of the inserted selection marker and replicon flanked by FRT sites was mediated by the FLP recombinase. By using the mentioned system, B. subtilis strain PT5 was constructed to harbor a genomic copy of the spac promoter-regulated 17 gene 1 located at wprA (encoding the cell wall-associated protease). Similarly, the 17 promoter-driven nattokinase or endoglucanase E1 of Thermomonospora fusca genes were also integrated into mpr (encoding an extracellular protease) of strain PT5. Consequently, the integrant PT5/Mmp-T7N or PT5/MT1-E1 resulted in a "clean" producer strain deprived of six proteases. After 24 h, the strain receiving induction was able to secret nattokinase and endoglucanase El with the volumetric activity reaching 10860 CU/mL and 8.4 U/mL, respectively. This result clearly indicates the great promise of the proposed approach for high secretion of recombinant proteins in B. subtilis.
URI: http://hdl.handle.net/11455/62053
ISSN: 0021-8561
文章連結: http://dx.doi.org/10.1021/jf100445a
顯示於類別:食品暨應用生物科技學系

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