title: Development of Pgrac100-based expression vectors allowing high protein production levels in Bacillus subtilis and relatively low basal expression in Escherichia coli.
abstract: BACKGROUND
  In general, fusion of recombinant genes to strong inducible promoters allowing intracellular expression in Bacillus subtilis is a two-step process. The ligation products are transformed into Escherichia coli, followed by identification of the correct plasmid, and this plasmid is subsequently transformed into B. subtilis. This raises the problem that basal level of expression of the recombinant gene could be harmful for E. coli cells. Based on the Pgrac promoter, we optimized the UP element, the -35, 15, -10 and the +1 region to enhance the promoter activity in B. subtilis after induction. However, detailed investigations for a promoter to develop expression vectors that allows high protein production levels in B. subtilis and a relatively low basal expression levels in E. coli has not been studied yet.
  
  
  RESULTS
  We screened the previously constructed library of E. coli - B. subtilis shuttle vectors for high level expression in B. subtilis and low basal level in E. coli. Promoter Pgrac100 turned out to meet these criteria, in which ß-galactosidase expression level of Pgrac100-bgaB is about 9.2 times higher than Pgrac01-bgaB in B. subtilis and the ratio of those in induced B. subtilis over un-induced E. coli from Pgrac100-bgaB is 1.3 times higher than Pgrac01-bgaB. Similarly, GFP expression level of Pgrac100-gfp is about 27 times higher than that of Pgrac01-gfp and the ratio from Pgrac100-gfp is 35.5 times higher than Pgrac01-gfp. This promoter was used as a basis for the construction of three novel vectors, pHT253 (His-tag-MCS), pHT254 (MCS-His-tag) and pHT255 (MCS-Strep-tag). Expression of the reporter proteins BgaB and GFP using these expression vectors in B. subtilis at a low IPTG concentration were measured and the fusion proteins could be purified easily in a single step by using Strep-Tactin or IMAC-Ni columns.
  
  
  CONCLUSIONS
  This paper describes the construction and analysis of an IPTG-inducible expression vector termed Pgrac100 for the high level production of intracellular recombinant proteins in B. subtilis and a relatively low basal expression level in E. coli. Based on this vector, the derivative vectors, Pgrac100-His-tag-MCS, Pgrac100-MCS-His-tag and Pgrac100-MCS-Strep-tag have been constructed.
authors.affiliation: Center for Bioscience and Biotechnology, VNUHCMC-University of Science, 227 Nguyen Van Cu District 5, Hochiminh, Vietnam. ptptrang@yahoo.com.|Laboratory of Molecular Biotechnology, VNUHCMC-University of Science, 227 Nguyen Van Cu District 5, Hochiminh, Vietnam. ptptrang@yahoo.com.
authors.forename: Trang Thi Phuong
authors.initials: TT
authors.lastname: Phan
authors.affiliation: Laboratory of Molecular Biotechnology, VNUHCMC-University of Science, 227 Nguyen Van Cu District 5, Hochiminh, Vietnam. tlthuoc@hcmus.edu.vn.
authors.forename: Linh Thuoc
authors.initials: LT
authors.lastname: Tran
authors.affiliation: Center for Bioscience and Biotechnology, VNUHCMC-University of Science, 227 Nguyen Van Cu District 5, Hochiminh, Vietnam. wschumann@uni-bayreuth.de.|Institute of Genetics, Universität of Bayreuth, Bayreuth, D-95440, Germany. wschumann@uni-bayreuth.de.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Schumann
authors.affiliation: Center for Bioscience and Biotechnology, VNUHCMC-University of Science, 227 Nguyen Van Cu District 5, Hochiminh, Vietnam. nguyen_hoang.xuatban@hotmail.com.|Laboratory of Molecular Biotechnology, VNUHCMC-University of Science, 227 Nguyen Van Cu District 5, Hochiminh, Vietnam. nguyen_hoang.xuatban@hotmail.com.|Department of Microbiology, VNUHCMC-University of Science, 227 Nguyen Van Cu District 5, Hochiminh, Vietnam. nguyen_hoang.xuatban@hotmail.com.
authors.forename: Hoang Duc
authors.initials: HD
authors.lastname: Nguyen
mesh_terms: D001412:Bacillus subtilis / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins