Development of Pgrac100-based expression vectors allowing high protein production levels in Bacillus subtilis and relatively low basal expression in Escherichia coli
© Phan et al.; licensee BioMed Central. 2015
Received: 7 April 2015
Accepted: 6 May 2015
Published: 21 May 2015
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.
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.
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.
The production of heterologous proteins in different microbial systems has revolutionized biotechnology. Most expression systems are based on an inducible promoter, and addition of the appropriate inducer leads to the production of the heterologous protein in most cases intracellularly. 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 [1–3].
Escherichia coli is the most widely used bacterial host to synthesize recombinant proteins for biochemical and functional studies. E. coli cells are easy to culture since they have a very short doubling time in rich media, and they are easy to manipulate genetically. 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].
Bacillus subtilis is an attractive alternative host for heterologous protein production and engineering because of the following reasons: (1) it can secrete proteins efficiently, especially homologous proteins up to 20 g/l; (2) it is nonpathogenic; (3) and it has been granted the GRAS (generally regarded as safe) status by the American Food and Drug Administration [6–8]. The authors have developed several plasmid-based expression vectors exhibiting structural stability , where induction can be accomplished by addition of xylose , IPTG , glycine  or by a cold shock .
Promoter P spac is one of the most-popular promoters used for expression of heterologous proteins in B. subtilis, but it is rather weak . The IPTG-inducible P grac promoter is 50 times stronger than P spac , and it has been derived from the B. subtilis groESL promoter and the E. coli lac operator [15, 16]. To further improve protein expression levels, we created a library of a second generation P grac promoters by either introducing promoter mutations in the consensus regions resulting in stronger promoters  or by applying the mRNA controllable stabilizing elements (CoSE) . However, enhancing the protein expression levels in B. subtilis also leads to higher basal expression levels in E. coli. In addition, some normal genes are not supposed to be harmful for E. coli, but it can inhibit the growth at high background expression levels, for examples ß-galactosidases, BgaB from Geobacillus stearothermophilus and LacZ from Escherichia coli . Therefore, expression vectors harboring promoters that control high protein production levels in B. subtilis after induction and allow a low basal level of expression in E. coli are of utmost importance. This study aims the development of expression vectors for B. subtilis based on a promoter that allows high inducible protein production in B. subtilis and relatively low basal level in E. coli.
Screening for an appropriate promoter
Choice of promoter Pgrac100
Expression of bgaB and gfp + under control of Pgrac01 and Pgrac100
7.7 ± 2.4
6.4 ± 1.4
5.3 ± 1.1
38.9 ± 5.8
50 ± 12
53 ± 15
257 ± 31
26 ± 9
75 ± 9
37 ± 5.7
222 ± 68
343 ± 78
489 ± 119
817 ± 79
68 ± 4.7
5 ± 0.7
47 ± 1.5
8 ± 0.5
7.0 ± 0.5
21 ± 0.6
190 ± 8
29 ± 0.7
73 ± 2.3
37 ± 2.6
63 ± 2.6
106 ± 9.4
568 ± 117
1554 ± 65
The results in Table 1 also showed that Pgrac100-bgaB seems to be characterized from high basal expression (222 ± 68 units) in B. subtilis. If low basal expression in E.coli is important to facilitate the cloning of toxic genes, the presence of basal expression in B. subtilis could make difficult the plasmid transformation. However, it also indicated that we could use Pgrac100 promoter for high production levels of recombinant protein at low concentration of IPTG inducer. If we consider low background expression levels in E. coli and B. subtilis, selection of Pgrac01  could be an option.
In comparison with other systems, we transformed PxylA-bgaB (pHCMC04-bgaB) and Pspac-bgaB (pHCMC05-bgaB)  into E. coli and spread transformants on X-gal plates, and the E. coli colonies developed blue color. Colonies of Pspac-bgaB were within the same range as those from Pgrac01-bgaB, while colonies from PxylA-bgaB were deeper blue than the others (Fig. 2c). When the E. coli cells were growth in liquid LB medium in the absence of the inducers, BgaB activities from Pspac-bgaB were equal to that of Pgrac01-bgaB, while those from PxylA-bgaB were within the same range as those from Pgrac100-bgaB (Fig. 2d). In B. subtilis, the BgaB expression levels of the two constructs, Pspac-bgaB and PxylA-bgaB in the presence of inducers were within the same range  and 50 times lower than Pgrac01-bgaB [15, 16]. Though Pspac expressed lower basal levels than and PxylA as high as Pgrac100 in E. coli, the expression levels in B. subtilis was also very low in the presence of inducer. Therefore, these promoters are not appropriate to be used for over-production of recombinant proteins in B. subtilis.
Important factors of Pgrac100 in controlling GFP expression
Bacterial strains, plasmids and oligonucleotides used in this study
E. coli OmniMAX
mcrA Δ(mrr hsdRMS-mcrBC); resistant to T1 and T5 phage; used for cloning
B. subtilis 1012
leuA8 metB5 trpC2 hsrM1
Pgrac01 (previously called Pgrac)
Pgrac100-8xHis-MCS (Start codon-His-tag-BamHI-XbaI-AatII-SmaI)
Pgrac100-MCS-His (BamHI-start codon-XbaI-AatII-His-tag-stop codon/TAA)
Pgrac100-MCS-Strep (BamHI-start codon-XbaI-AatII-Strep-tag-stop codon/TAA)
From Nguyen H. N
From Nguyen H. N
Sequence 5′ → 3′
pHT1179 and pHT1180
pHT1179 and pHT1180
pHT1169 and pHT1171
pHT100-gfp + and pHT1169
The expression levels of GFP of Pgrac100-gfp increased after addition of IPTG and reached up to 568 RFU at 0.01 mM IPTG, 27-fold higher than that of Pgrac01-gfp (Table 1) and 4.7-fold higher than that of Pgrac212-gfp. In addition, we calculated the induction factor and the ratio of the activities of induced and un-induced samples. Pgrac100 exhibited an induction factor of 9 at 0.01 mM IPTG and of 25 at 0.1 mM IPTG (Table 1), while those of Pgrac01 were 2.6 and 24.7, respectively (Table 1) and those of Pgrac212 6.3 and 77 (data not shown). Similar results using BgaB as reporter were also observed for Pgrac100 (Table 1). The substantial differences in protein expression levels between BgaB and GFP might be because they come from two different organisms, BgaB from G. stearothermophilus and GFP from Aequorea victoria, and the sequences of the genes might influence the transcription and/or translation efficiency in E. coli and B. subtilis. These results demonstrate that Pgrac100 not only tightly controls the background expression level in E. coli, but also allowed high protein production levels at low IPTG concentrations. In summary, promoter Pgrac100 is an excellent choice for the construction of inducible expression vectors for B. subtilis.
Construction of basic expression vectors
The above result demonstrated that promoter Pgrac100 allowed high protein production levels in B. subtilis and low background expression levels in E. coli by using two reporter proteins, BgaB and GFP. To generate tagging expression vectors, we removed bgaB from pHT100  and added the DNA fragments containing start codon-His-tag-BamHI-XbaI-AatII-SmaI, BamHI-start codon-XbaI-AatII-His-tag-stop codon/TAA or BamHI-start codon-XbaI-AatII-Strep-tag-stop codon/TAA, resulting pHT253, pHT254, and pHT255, respectively (Table 2). Fig. 1c shows the DNA sequence of the multi-cloning site, the His-tag, the start and the stop codon from pHT254. The other plasmids were adapted appropriately to meet these requirements. The full sequences of these three plasmids were similar to pHT01  except for the promoter regions and the multi-cloning sites with different tags. The map of plasmid pHT254 is shown in Fig. 1a. Fig. 2b indicates the differences between promoter Pgrac01 and Pgrac100 at the UP element and the −35 and −15 regions. Target genes can be introduced using restriction enzymes BamHI, XbaI, AatII or SmaI as fusions or non-fusions with either a His-tag or a Strep-tag at the N- or C-terminus.
Evaluation of the expression vectors in B. subtilis
The fusions, BgaB-His (Fig. 3b), BgaB-Strep (Fig. 3c), GFP-His (Fig. 3e) and GFP-Strep (Fig. 3f) are produced at levels comparable to those without a purification tag, BgaB (from pHT100-bgaB) and GFP (from pHT100-gfp) deduced from SDS-PAGE gels. The BgaB expression levels could reach up to 30 % of total cellular proteins , while the tagged versions accumulated 24 % using 0.1 mM IPTG (Fig. 3b and c) and up to 30 % using 1 mM IPTG. Similarly, the untagged and the C-tagged versions of GFP could be produced at 15 % of total cellular proteins on the average (Figure 3e and f). However, the expression levels of the fusions at low concentrations of IPTG were lower than the untagged constructs. Besides in B. subtilis 1012, we also checked the expression in B. subtilis WB800N , a derivative of WB800 , a derivative of strain 168. The expression levels in WB800N were similar to those in 1012 (data not shown). These results indicate that the expression vectors pHT253, pHT254, pHT255 could be used for overproduction of recombinant proteins to high levels in different B. subtilis strains.
We show that the artificial promoter Pgrac100 could be used for the construction of His- or Strep-tagged versions, pHT253, pHT254 and pHT255 for B. subtilis. These three new expression vectors provide two advantages: (i) allowing high production levels of recombinant proteins in B. subtilis after induction and (ii) maintaining relatively low background expression levels in E. coli.
Bacterial strains, plasmids and growth conditions
E. coli strain OmniMAX (Invitrogen) was used as the recipient in all cloning experiments and to determine the expression levels. B. subtilis strains, 1012  and WB800N  were used to analyze expression of the bgaB and gfp+ genes. A list of the plasmids and oligonucleotides used in this study is shown in Table 2. Cells were routinely grown in Luria broth (LB) at 37 °C under shaking at 200 rpm. Antibiotics were added where appropriate (ampicillin at 100 μg/mL for E. coli and chloramphenicol at 10 μg/mL for B. subtilis).
Construction of plasmids
The plasmid pHT100  carrying promoter Pgrac100 fused to the reporter gene bgaB was used as backbone. To generate the primary expression vectors, we removed the bgaB gene and inserted DNA sequences coding for a His- or a Strep-tag either to the N- or to the C-terminus. Three pairs of complementary oligonucleotides (ON), ON301F and ON302R, ON303F and ON304R, ON305F and ON306R were used for these purposes. The complementary mixtures were ligated with the BamHI- and AatII-treated pHT100 resulting in pHT253, pHT254 and pHT255. These vectors contain start codon-His-tag-BamHI-XbaI-AatII-SmaI (pHT253), BamHI-start codon-XbaI-AatII-His-tag-stop codon/TAA (pHT254) or BamHI-start codon-XbaI-AatII-Strep-tag-stop codon/TAA (pHT255). To construct pHT100-gfp + (GFP+), pHT1169 (His-tag-GFP+) and pHT1171 (GFP + −Strep-tag), we amplified the gfp + gene using the primer pairs, ON1279 and ON1280 for pHT100-gfp+, ON1277and ON1280 for pHT1169, and ON1277 and ON1278 for pHT1171 with pHT10-gfp + . The BglII- or BamHI- and AatII-treated PCR products were introduced into pHT100, pHT253 or pHT255 at BamHI and AatII, resulting in pHT100-gfp+, pHT1169 and pHT1171, respectively. To construct pHT1179 (bgaB-His-tag) and pHT1180 (bgaB-Strep-tag), we amplified the bgaB gene using primers ON941 and ON1250 with pNDH33-bgaB  as template. The BamHI- and AatII-treated PCR products were ligated into pHT254 and pHT255 at their BamHI and AatII sites resulting in pHT1179 and pHT1180, respectively.
Measurement of the BgaB and GFP production levels in E. coli and in B. subtilis
Three colonies were cultured in 0.5 ml LB medium containing the appropriate antibiotic in a 96 well-block (Eppendorf block) and shaken overnight at 200 rpm at room temperature (25 °C). The pre-culture of each clone (75 μl) was transferred to 3 ml LB medium containing the appropriate antibiotic in a 24-well-block. The block was incubated at 37 °C with shaking at 200 rpm. When the OD600 of the culture reached 0.6 – 1, the cells were induced by addition of IPTG at final concentration of 0 mM, 0.001 mM, 0.01 mM and 0.1 mM. The cells were harvested after 2 or 4 h of induction. The cells were collected in Eppendorf tubes at an OD600 of 2.5 after centrifugation. Samples were prepared for activity measurements or SDS-PAGE. The cells were lysed by lysozyme and sample buffer was added to 150 μl, and 8 μl each were applied to SDS-PAGE. ß-galactosidase activities were measured as described . For E. coli, the GFP cells were re-suspended in 300 μl BPS, 12 μl chloroform, and 6 μl SDS 0.1 % were added followed by shaking for 1 h. For B. subtilis, the GFP cells were lysed in 300 μl PBS containing 1 mg/ml lysozyme and incubated at 37 °C for 2 h. The samples were centrifuged at 10 000 rpm for 5 min and used for determination of the activities. GFP activities were measured by using a Synergy HT Multi-mode Microplate Reader and 384 W plate (Black) with an excitation wavelength at 485 (+/−20) nm and an emission wavelength at 520 (+/−20) nm. The experiment was carried with at least three different colonies, and standard errors were calculated.
Affinity purification of the fusion proteins
B. subtilis 1012 carrying different plasmids were grown in LB medium to mid-log phase, and production of the recombinant proteins was induced by addition of 0.1 mM IPTG. The cells were collected by centrifugation and re-suspended in the desired buffers with lysozyme (0.25 mg/ml) and disrupted by sonification. For His-tag fusion proteins, the protocol with recommended buffers for Ni-NTA Spin Columns (Qiagen) was applied, in which the washing buffer contain 40 mM imidazole. For Strep-tag fusion proteins, the Strep-Tactin Spin column kit (IBA) was used.
This research was partially funded by the Vietnam National Foundation for Science and Technology Development (Nafosted) under grant number 106.16-2011.80. The equipment was supported by TWAS under research grants 11–188 /RG/BIO/AS_G.
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