Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/68252
標題: Construction and characterization of nisin-controlled expression vectors for use in Lactobacillus reuteri
作者: Wu, C.M.
Lin, C.F.
Chang, Y.C.
Chung, R.C.
關鍵字: Lactobacillus reuteri
nisin-controlled expression system
signal
peptide
expression-secretion vector
alpha-amylase
controlled gene-expression
lactic-acid bacteria
alpha-amylase
lactococcus-lactis
secretion signals
bacillus-subtilis
escherichia-coli
cloning vectors
induction
proteins
期刊/報告no:: Bioscience Biotechnology and Biochemistry, Volume 70, Issue 4, Page(s) 757-767.
摘要: The Nisin-controlled gene expression (NICE) system, which was discovered in Lactococcus lactis, was adapted to Lactobacillus reuteri by ligating nisA promoter (PnisA) and nisRK DNA fragments into the Escherichia coli-Lb. reuteri shuttle vector pSTE32. This chimerical plasmid (pNICE) was capable of expressing the heterologous amylase gene (amyL) under nisin induction., Optimization of induction factors for this Lb. reuteri/pNICE system, including nisin concentration (viz. 50 ng/ml), growth phase of culture at which nisin be added (viz. at the early exponential phase), and the best time for analyzing the gene product after inoculation (viz. at the 3rd h), allowed the amylase product to be expressed in high amounts, constituting up to about 18% of the total intracellular protein. Furthermore, the signal peptide (SP) of amyL gene (SPamyL) from Bacillus licheniformis was ligated to the downstream of PnisA in pNICE, upgrading this vector to a NICE-secretion (NIES) level, which was then designated pNIES (Sec(+), secretion positive). Characterization of pNIES using an amyL-SP Delta gene (amyL gene lacking its SP) as a reporter revealed the 3rd h after induction as the secretion peak of this system, at which the secretion efficiency and the amount of alpha-amylase being secreted into the culture supernatant were estimated to reach 77.6% and 27.75 mg/l. Expression and secretion of AmyL products by pNIES in Lb. reuteri was also confirmed by SDS-PAGE and immunoblotting analysis.
URI: http://hdl.handle.net/11455/68252
ISSN: 0916-8451
文章連結: http://dx.doi.org/10.1271/bbb.70.757
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