Abstract
BackgroundIn 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.ResultsWe 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.ConclusionsThis 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.
Highlights
Fusion of recombinant genes to strong inducible promoters allowing intracellular expression in Bacillus subtilis is a two-step process
Identification of a suitable inducible promoter controlling high production levels of recombinant proteins in B. subtilis and, at the same time, retaining relatively low basal levels in E. coli in the absence of the inducer is an important requirement during construction of expression vectors for B. subtilis
We used the Pgrac-promoter library described [11, 17] and screened for low BgaB expression in E. coli by using the method described for B. subtilis [18]
Summary
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. 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. Microbial expression systems have been described for bacteria, yeast, filamentous fungi, and unicellular algae. All these systems have advantages and disadvantages, which have been extensively discussed [1,2,3]. Three disadvantages related to E. coli are: (1) low expression of some heterologous genes; (2) some heterologous proteins are insoluble and form inclusion bodies; and (3) contamination of the heterologous proteins by the endotoxin LPS [4, 5]
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