Construction and Characterization of Nisin-Controlled Expression Vectors for Use in Lactobacillus reuteri
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概要
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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-SPΔ 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 α-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.
- 社団法人 日本農芸化学会の論文
- 2006-04-23
著者
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Wu Chi-Ming
Graduate Institute of Veterinary Pathobiology, College of Veterinary Medicine, National Chung Hsing
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Wu Chi-ming
Graduate Inst. Of Veterinary Pathobiology Coll. Of Veterinary Medicine National Chung Hsing Univ.
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Chung Tung-ching
Department Of Veterinary Medicine National Chung Hsing University
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LIN Chuen-Fu
Department of Veterinary Medicine, National Chung-Hsing University
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Lin Chuen-fu
Department Of Medical Technology Central Taiwan University Of Science And Technology
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Wu Chi-ming
Graduate Institute Of Veterinary Microbiology National Chung-hsing University
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CHANG Yi-Chih
Department of Medical Laboratory Science and Biotechnology, China Medical University
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Chang Yi-chih
Department Of Medical Laboratory Science And Biotechnology China Medical University
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Chung Tung-ching
Department Of Veterinary Medicine National Chung-hsing University
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LIN Chuen-Fu
Department of Medical Laboratory Science and Biotechnology, Central Taiwan University of Science and Technology
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