N-terminal LysSN-His-tag improves the production of intracellular recombinant protein in Bacillus subtilis.

Choosing fusion tags to enhance the recombinant protein levels in the cytoplasm of Bacillus subtilis has been limited. Our previous study demonstrated that His-tag at the N-terminus could increase the expression levels of the low-expression gene egfp, while significantly reducing the high-expression genes gfp+ and bgaB in the cytoplasm of B. subtilis. In this study, we aimed to prove the potential of a fusion tag, the combination of the N-terminal domain of B. subtilis lysyl tRNA synthetase (LysSN) and His-tag with varying numbers of histidine (6xHis, 8xHis, 10xHis) by investigating their effects on the expression levels of egfp, gfp+ and bgaB in B. subtilis. For the low-expression gene, LysSN-xHis-tag could enhance the fluorescent intensity of EGFP 23.5 times higher than EGFP without a fusion tag, and 1.5 times higher than that fused with only His-tag. For high-expression genes, the expression level of BgaB and GFP+ was 2.9 and 12.5 times higher than that of His-tag, respectively. The number of histidines in LysSN-xHis-tag did not influence the expression levels of the high-expression genes but affected the expression levels of the low-expression gene.

Potent IPTG-inducible integrative expression vectors for production of recombinant proteins in Bacillus subtilis.

The IPTG-inducible promoter family, Pgrac, allows high protein expression levels in an inducible manner. In this study, we constructed IPTG-inducible expression vectors containing strong Pgrac promoters that allow integration of the transgene at either the amyE or lacA locus or both loci in Bacillus subtilis. Our novel integrative expression vectors based on Pgrac promoters could control the repression of protein production in the absence and the induction in the presence of an inducer, IPTG. The ß-galactosidase (BgaB) protein levels were 9.0%, 15% and 30% of the total cellular protein in the B. subtilis strains carrying single cassettes with the Pgrac01, Pgrac100 or Pgrac212 promoters, respectively. The maximal induction ratio of Pgrac01-bgaB was 35.5 while that of Pgrac100-bgaB was 7.5 and that of Pgrac212-bgaB was 9. The inducible expression of GFP and BgaB protein was stably maintained for 24 h, with the highest yield of GFP being 24% of cell total protein while the maximum amount of BgaB was found to be 38%. A dual integration of two copies of the gfp+ gene into the B. subtilis genome at the lacA and amyE loci resulted in a yield of about 40% of total cellular protein and a 1.74-fold increase in GFP compared with single-integrated strains containing the same Pgrac212 promoter. The capability of protein production from low to high levels of these inducible integrative systems is useful for fundamental and applied research in B. subtilis.

Influence of N-terminal His-tags on the production of recombinant proteins in the cytoplasm of Bacillus subtilis.

The influence of fusion tags to produce recombinant proteins in the cytoplasm of Bacillus subtilis is not well-studied as in E. coli. This study aimed to investigate the influence of His-tags with different codons on the protein production levels of the high expression gene (gfp+) and low expression gene (egfp) in the cytoplasm of B. subtilis cells. We used three different N-terminal His-tags, M-6xHis, MRGS-8xHis and MEA-8xHis, to investigate their effects on the production levels of GFP variants under the control of the Pgrac212 in B. subtilis. The fusions of His-tags with GFP+ caused a reduction compared to the construct without His-tag. When three His-tags fused with egfp, the EGFP production levels were significantly increased up to 3.5-, 12-, and 15-fold. This study suggested that His-tag at the N-terminus could enhance the protein production for the low expression gene and reduce that of the high expression gene in B. subtilis.

Integrative expression vectors with Pgrac promoters for inducer-free overproduction of recombinant proteins in Bacillus subtilis.

Inducer-free integrative vectors are often used to create B. subtilis strains for industrial purposes, but employing strong promoters to produce high levels of recombinant proteins in B. subtilis results in high leaky expression that can hamper cloning in Escherichia coli. To overcome the problem, we used strong IPTG-inducible Pgrac promoters harboring lac operators to construct inducer-free integrative vectors able to integrate into the B. subtilis genome at either the lacA or the amyE locus, or both and examined their ability to repress the ß-galactosidase (bgaB) gene in E. coli and to overexpress BgaB in B. subtilis. The Pgrac01 vectors could repress bgaB expression about 24-fold in E. coli to low background levels. The integrated Pgrac01-bgaB constructs exhibited inducer-free expression and produced 8% of total cellular proteins, only 1.25 or 1.75 times less compared with their cognates as plasmids. The stronger promoters, Pgrac100-bgaB and Pgrac212-bgaB yielded 20.9 % and 42 % of total intracellular proteins after 12 h of incubation, respectively. Incorporation of the Pgrac212-bgaB into both amyE and lacA loci resulted in BgaB expression up to 53.4 %. In conclusion, integrative vectors containing the Pgrac promoter family have great potential for inducer-free overproduction of recombinant proteins in B. subtilis.

Using the IPTG-Inducible Pgrac212 Promoter for Overexpression of Human Rhinovirus 3C Protease Fusions in the Cytoplasm of Bacillus subtilis Cells.

Expression and secretion of recombinant proteins in the endotoxin-free bacterium, Bacillus subtilis, has been thoroughly studied, but overexpression in the cytoplasm has been limited to only a few proteins. Here, we used the robust IPTG-inducible promoter, Pgrac212, to overexpress human rhinovirus 3C protease (HRV3C) in the cytoplasm of B. subtilis cells. A novel solubility tag, the N-terminal domain of the lysS gene of B. subtilis coding for a lysyl-tRNA synthetase was placed at the N terminus with a cleavage site for the endoprotease HRV3C, followed by His-HRV3C or His-GST-HRV3C. The recombinant protease was purified by using a Ni-NTA column. In this study, the His-HRV3C and His-GST-HRV3C proteases were overexpressed in the cytoplasm of B. subtilis at 11% and 16% of the total cellular proteins, respectively. The specific protease activities were 8065 U/mg for His-HRV3C and 3623 U/mg for His-GST-HRV3C. The purified enzymes were used to cleave two different substrates followed by purification of the two different protein targets, the green fluorescent protein and the beta-galactosidase. In conclusion, the combination of an inducible promoter Pgrac212 and a solubility tag allowed the overexpression of the HRV3C protease in the cytoplasm of B. subtilis. The resulting fusion protein was purified using a nickel column and was active in cleaving target proteins to remove the fusion tags. This study offers an effective method for producing recombinant proteins in the cytoplasm of endotoxin-free bacteria.

Development of inducer-free expression plasmids based on IPTG-inducible promoters for Bacillus subtilis.

BACKGROUND: Besides Escherichia coli, Bacillus subtilis is an important bacterial species for the production of recombinant proteins. Recombinant genes are inserted into shuttle expression vectors which replicate in both E. coli and in B. subtilis. The ligation products are first transformed into E. coli cells, analyzed for correct insertions, and the correct recombinant plasmids are then transformed into B. subtilis. A major problem using E. coli cells can be the strong basal level of expression of the recombinant protein which may interfere with the stability of the cells. To minimize this problem, we developed strong expression vectors being repressed in E. coli and inducer-free in B. subtilis. RESULTS: In general, induction of IPTG-inducible expression vectors is determined by the regulatory lacI gene encoding the LacI repressor in combination with the lacO operator on the promoter. To investigate the inducer-free properties of the vectors, we constructed inducer-free expression plasmids by removing the lacI gene and characterized their properties. First, we examined the ability to repress a reporter gene in E. coli, which is a prominent property facilitating the construction of the expression vectors carrying a target gene. The ß-galactosidase (bgaB gene) basal levels expressed from Pgrac01-bgaB could be repressed at least twice in the E. coli cloning strain. Second, the inducer-free production of BgaB from four different plasmids with the Pgrac01 promoter in B. subtilis was investigated. As expected, BgaB expression levels of inducer-free constructs are at least 37 times higher than that of the inducible constructs in the absence of IPTG, and comparable to those in the presence of the inducer. Third, using efficient IPTG-inducible expression vectors containing the strong promoter Pgrac100, we could convert them into inducer-free expression plasmids. The BgaB production levels from the inducer-free plasmid in the absence of the inducer were at least 4.5 times higher than that of the inducible vector using the same promoter. Finally, we used gfp as a reporter gene in combination with the two promoters Pgrac01 and Pgrac100 to test the new vector types. The GFP expression levels could be repressed at least 1.5 times for the Pgrac01-gfp+ inducer-free construct in E. coli. The inducer-free constructs Pgrac01-gfp+ and Pgrac100-gfp+ allowed GFP expression at high levels from 23 × 104 to 32 × 104 RFU units and 9-13% of total intracellular proteins. We could reconfirm the two major advantages of the new inducer-free expression plasmids: (1) Strong repression of the target gene expression in the E. coli cloning strain, and (2) production of the target protein at high levels in B. subtilis in the absence of the inducer. CONCLUSIONS: We propose a general strategy to generate inducer-free expression vector by using IPTG-inducible vectors, and more specifically we developed inducer-free expression plasmids using IPTG-inducible promoters in the absence of the LacI repressor. These plasmids could be an excellent choice for high-level production of recombinant proteins in B. subtilis without the addition of inducer and at the same time maintaining a low basal level of the recombinant proteins in E. coli. The repression of the recombinant gene expression would facilitate cloning of genes that potentially inhibit the growth of E. coli cloning strains. The inducer-free expression plasmids will be extended versions of the current available IPTG-inducible expression vectors for B. subtilis, in which all these vectors use the same cognate promoters. These inducer-free and previously developed IPTG-inducible expression plasmids will be a useful cassette to study gene expression at a small scale up to a larger scale up for the production of recombinant proteins.

Regulation of bacterial heat shock stimulons.

All organisms developed genetic programs to allow their survival under stressful conditions. In most cases, they increase the amount of a specific class of proteins which deal with the stress factor and allow cells to adapt to life-threatening conditions. One class of stress proteins are the heat shock proteins (HSPs) the amount of which is significantly increased after a sudden temperature rise. How is the heat shock response (HSR) regulated in bacteria? This has been studied in detail in Escherichia coli, Bacillus subtilis and Streptomyces spp. Two major mechanisms have been described so far to regulate expression of the HSGs, namely alternative sigma factors and transcriptional repressors. This review focuses on the regulatory details of the different heat shock regulons in the three well-studied bacterial species.

Development of Pgrac100-based expression vectors allowing high protein production levels in Bacillus subtilis and relatively low basal expression in Escherichia coli.

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.

Disulfide Bonds of Proteins Displayed on Spores of Bacillus subtilis Can Occur Spontaneously.

Surface display using spores of Bacillus subtilis is widely used to anchor antigens and enzymes of different sources. One open question is whether anchored proteins are able to form disulfide bonds. To answer this important question, we anchored the Escherichia coli alkaline phosphatase PhoA on the spore surface using two different surface proteins, CotB and CotZ. This enzyme needs two disulfide bonds to become active. Subsequently, we purified the spores and assayed for alkaline phosphatase activity. In both cases, we were able to recover enzymatic activity. Next, we asked whether formation of disulfide bonds occurs spontaneous or is catalyzed by thiol-disulfide oxidoreductases upon lysis of the cells. The experiment was repeated in a double-knockout mutant ΔbdbC and ΔbdbD. Since the disulfide bonds are also present on spores prepared from the double knockout, we conclude that oxidative environment after cell lysis is sufficient for disulfide formation of alkaline phosphatase.

Use of IPTG-inducible promoters for anchoring recombinant proteins on the Bacillus subtilis spore surface.

The method of surface display allows the fusion of passenger proteins to a carrier protein displayed on the outside of bioparticles such as spores. Here, we used spores of Bacillus subtilis, the outer surface proteins CotB, CotC, and CotG as carrier and the amyQ-encoded α-amylase and GFPuv as passenger proteins. The different translational fusions were fused to two different IPTG-inducible promoters, and the regulated expression level of both passenger proteins were measured in relation to the inducer concentration added to sporulating cells. It turned out that the amount of fusion protein on the outside of spores was dependent on the amount of IPTG added, but the optimal amount of inducer varied depending on the carrier and the passenger proteins. These experiments demonstrate that a regulatable expression of passenger proteins on the surface of spores is possible. This will help to adjust the amount of any passenger protein to that needed for specific purposes.

Construction and analysis of a modified transposable element carrying an outward directed inducible promoter for Bacillus subtilis.

Transposons are important tools to inactivate chromosomal genes followed by a correlation with a particular phenotype or genotype. Here we demonstrated the development of a special type of genetically engineered transposon carrying an outward-directed inducible promoter in order to allow transcription of nearby genes. We have modified the mariner transposon TnYLB able to transpose in B. subtilis. This modified TnYLB carries an expression unit consisting of the xylose repressor xylR and an outward-directed promoter negatively controlled by this repressor. This TnYLB-XylOut transposon is able to turn on gene expression if insertion occurs close to a promoter-less gene. It will be an important tool to identify the function of genes either by turning on their expression or by enhanced expression depending on the xylose concentration.

Construction of a 5'-controllable stabilizing element (CoSE) for over-production of heterologous proteins at high levels in Bacillus subtilis.

Different mRNA stabilizing elements including the 3'-stem-loop, the ribosome binding sites (RBS), the 5'-stem-loop and the spacer region between the RBS and the 5'-stem-loop were analysed in detail to increase mRNA stability resulting in enhanced expression of heterologous proteins. In addition, in combination with mRNA stabilizing elements, we propose a new class of 5'-mRNA controllable stabilizing element (CoSE) which is composed of a transcriptional operator such as lacO of the Escherichia coli lac operon and a suitable RBS followed by an optimal spacer length. Such a CoSE allowed Bacillus subtilis cells to synthesize extraordinary stable transcripts with a half-life of the model bgaB reporter transcript (codes for an ß-galactosidase gene derived from Bacillus stearothermophilus) of more than 60 min. This CoSE will be an important tool to control mRNA stability in cells for both research and biotechnological applications. For example, this CoSE can be used in inducible expression vectors offering two major advantages: (i) controlling transcription of target genes by the inducer and (ii) enhancing the stability of the transcript allowing the production high levels of recombinant proteins.

Construction of an artificial secYEG operon allowing high level secretion of α-amylase.

The key components of the major secretion pathway in bacteria, the Sec pathway, are the proteins SecA, an ATPase that generates the energy required for protein translocation, and the heterotrimeric protein complex SecYEG, which functions as the preprotein-conducting channel through the cytoplasmic membrane, named translocon. Overexpression of exoproteins can cause jamming of the membrane, e.g., due to a shortage of translocons. Therefore, we decided to increase the number of translocons by first creating an artificial secYEG operon and then fusing it to an inducible promoter. By Western- and Northern-blot analysis, we could first show that the amount of the SecY protein and the secYEG transcript can be increased after addition of the inducer. Next, we proved by immunoblot experiments that the amount of α-amylase secreted in the presence of increased amounts of SecYEG proteins is enhanced. Therefore, increasing the number of translocons is accompanied by a concomitant increase in the amount exoenzymes. This finding will be of importance for high-level secretion of recombinant proteins.

Inactivation Effect of Standard and Fractionated Electron Beam Irradiation on Enveloped and Non-Enveloped Viruses in a Tendon Transplant Model.

BACKGROUND: For increasing allograft tendon safety in reconstructive surgery, an effective sterilization method achieving sterility assurance including viruses without impairing the grafts properties is needed. Fractionated Electron Beam (Ebeam) has shown promising in vitro results. The proof of sufficient virus inactivation is a central part of the process validation. METHODS: The Ebeam irradiation of the investigated viruses was performed in an optimized manner (oxygen content < 0.1%, -78 °C). Using principles of a tendon model the virus inactivation kinetics for HIV-2, HAV, pseudorabies virus (PRV) and porcine parvovirus (PPV) were calculated as TCID(50)/ml and D(10) value (kGy) for the fractionated (10 × 3.4 kGy) and the standard (1 × 34 kGy) Ebeam irradiation. RESULTS: All viruses showed comparable D(10) values for both Ebeam treatments. For sufficient virus titer reduction of 4 log(10) TCID(50)/ml, a dose of 34 kGy of the fractionated Ebeam irradiation was necessary in case of HIV-2, which was the most resistant virus investigated in this study. CONCLUSION: The fractionated and the standard Ebeam irradiation procedure revealed comparable and sufficient virus inactivation capacities. In combination with the known good biomechanical properties of fractionated Ebeam irradiated tendons, this method could be a safe and effective option for the terminal sterilization of soft tissue allografts.

Thermosensor systems in eubacteria.

Four different mechanisms have evolved in eubacteria to comply with changes in the environmental temperature. The underlying genetic mechanisms regulate gene expression at transcriptional, translational and posttranslational level. The high temperature response (HTR) is a reaction on increases in temperature and is mainly used by pathogenic bacteria when they enter their mammalian host. The temperature of 37°C causes induction of the virulent genes the products of which are only needed in this environment. The heat shock response (HSR) is induced by any sudden increase in temperature, allows the bacterial cell to adapt to this environmental stress factor and is shut off after adaptation. In a similar way the low temperature response (LTR) is a reaction to a new environment and leads to the constant expression of appropriate genes. In contrast, the cold shock response (CSR) includes turn off of the cold shock genes after adaptation to the low temperature. Sensors of temperature changes are specific DNA regions, RNA molecules or proteins and conformational changes have been identified as a common motif.

Development of a strong intracellular expression system for Bacillus subtilis by optimizing promoter elements.

Transcription efficiency of inducible promoters remains a bottleneck in recombinant protein production in Bacillus subtilis cells. Here, we present experimental data how to generate strong IPTG-inducible promoters by optimization of nucleotides at the conserved regions of the groESL promoter including the UP element, the -35, -15, -10 and the +1 region. Combination of these changes into one promoter enhanced the amount of recombinant proteins accumulating intracellularly up to about 30% of the total cellular protein.

The sporulation control gene spo0M of Bacillus subtilis is a target of the FtsH metalloprotease.

In an attempt to identify substrate proteins of the FtsH metalloprotease involved in stage 0 of sporulation in Bacillus subtilis, the proteome of an ftsH wild-type strain was compared to that of an ftsH null mutant by the 2D gel technique. One of the most abundant proteins identified in the absence of ftsH turned out to be Spo0M, a sporulation control protein of stage 0. Using transcriptional fusion between the spo0M promoter and the bgaB reporter gene, expression analysis did not reveal any influence of FtsH on spo0M transcription, suggesting that FtsH might have a negative effect on the stability of Spo0M due to its proteolytic activity on Spo0M. Indeed, in vitro incubation of purified components demonstrated that Spo0M was degraded by FtsH, indicating that it serves as a substrate for the FtsH protease. These results are discussed.

Analysis and application of Bacillus subtilis sortases to anchor recombinant proteins on the cell wall.

Bacillus subtilis codes for two putative sortases, YhcS and YwpE, and two surface proteins, YhcR and YfkN, harboring sorting motifs supposed to be recognized by the putative sortase(s). However, there is no experimental evidence to show a direct link between these sortases and sorting sequences. To study the role of these two putative sortases on displaying YhcR and YfkN on the cell wall, expression of yhcS and ywpE was analyzed by transcriptional fusions and by Northern blot. It turned out that yhcS gene is expressed at a higher level during the late stationary phase from both experiments, while ywpE expression is not confirmed in the Northern blot analysis. Next, we constructed yhcS and ywpE single and double knockout strains and plasmids that express one or both genes to restore the functions of the knockout strains. It could be shown that display of YhcR and YfkN on the surface depended on the presence of YhcS while YwpE seems not to play a major role if any as a sortase. Finally, the putative sorting motif together with a 123-amino-acid spacer derived from YhcR and YfkN designated YhcR123 and YfkN123, respectively, were fused to an α-amylase reporter enzyme. The fusion protein YhcR123-AmyQ could be displayed on the surface at high amounts, while YfkN123-AmyQ could be hardly detected. We conclude that the sortase YhcS can recognize and anchor YhcR on the cell wall. This result further indicates that the YhcR sorting sequence can be used to display recombinant proteins on the surface of B. subtilis cells.

Establishment of a simple and rapid method to screen for strong promoters in Bacillus subtilis.

Using published plasmid vectors containing the bgaB gene encoding a heat-stable beta-galactosidase, we have been unable to fuse strong promoters to this reporter gene. In addition, we could not analyze the promoter strength by a plate assay. Therefore, we constructed an Escherichia coli-Bacillus subtilis shuttle vector to allow the cloning of strong promoters and their rapid analysis in B. subtilis by plating on X-Gal plates in the presence of the inducer IPTG. We show that the blue color of the colonies reflects the strength of the promoters, which was verified by measuring the beta-galactosidase activities.

Transcriptional analysis of the lysine-responsive and riboswitch-regulated lysC gene of Bacillus subtilis.

About 2% of the Bacillus subtilis genes are subject to regulation by riboswitch-controlled mechanisms. One of them is the L-lysine-dependent lysC gene which is turned on when the L-lysine concentration within the cytoplasm is low. In the presence of a high L-lysine concentration, only a 0.27-kb transcript is synthesized representing the riboswitch due to transcription termination. When the L-lysine concentration is low, the full-length 1.6-kb transcript is produced due to transcription anti-termination. Here, we show for the first time that even under conditions of transcription anti-termination the truncated form of the RNA is still predominant. This 0.27-kb transcript is neither the result of enhanced stability nor does it result from processing of the full-length transcript. When the region coding for the transcription terminator was removed, the riboswitch RNA failed to be produced. These data were confirmed by analysis of a transcriptional fusion between the promoter-riboswitch region of lysC with and without a functional transcriptional terminator and the lacZ reporter gene. The putative function(s) of the riboswitch under conditions of low L-lysine concentration is discussed.