Plasmid 54 (2005) 241–248 www.elsevier.com/locate/yplas 0147-619X/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.plasmid.2005.05.001 Construction of plasmid-based expression vectors for Bacillus subtilis exhibiting full structural stability Hoang Duc Nguyen a,b , Quynh Anh Nguyen b , Rita C. Ferreira c , Luis C.S. Ferreira c , Linh Thuoc Tran b , Wolfgang Schumann a,¤ a Institute of Genetics, University of Bayreuth, D-95440 Bayreuth, Germany b Vietnam National University-Ho Chi Minh City, College of Natural Sciences, Faculty of Biology, Ho Chi Minh City, Viet Nam c Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brazil Received 8 February 2005, revised 4 May 2005 Available online 11 July 2005 Communicated by Margarita Salas Abstract A series of plasmid-based expression vectors have been constructed allowing stable intracellular expression of recom- binant proteins in Bacillus subtilis strains. These expression vectors are based on the recently described Escherichia coli– B. subtilis shuttle vector pMTLBS72 which replicates as theta circles. Besides the weak constitutive promoter P lepA , we inserted three diVerent controllable promoters: P gsiB which can be induced by heat and acid shock, and by ethanol, P xylA and P spac which respond to the addition of xylose and IPTG, respectively. The versatility of these expression vectors was demonstrated by fusing their promoters to a reporter gene and by overexpression of the HtpG protein with three of them. All recombinant vectors exhibited full structural stability.  2005 Elsevier Inc. All rights reserved. Keywords: Promoters lepA, gsiB, xylA, and spac; htpG 1. Introduction High-level production of recombinant proteins is a prerequisite for their subsequent puri Wcation. In most cases for the production of heterologous pro- teins, Escherichia coli cells are used as a protein fac- tory (see recent review article by Schumann and Ferreira, 2004). Attempts to develop Bacillus subtilis as a second protein factory where the recombinant proteins are secreted into the medium have often not been successful because of two major reasons: (i) structural instability of the recombinant plasmids * Corresponding author. Fax: +49 921 552710. E-mail address: wschumann@uni-bayreuth.de (W. Schumann).