Polycistronic expression of CGA-N 46 gene in Bacillus subtilis DB 1342

CGA-N46 is a small antifungal derived peptide and consists of the 31st to 76th amino acids of the Nterminus of human Chromogranin A. Polycistronic expression of CGA-N46 gene in Bacillus subtilis DB1342 was used to improve its production. Single, double and three copies of exogenous gene fragment which contained ribosome binding site (RBS), sacB signal peptide nucleotide sequence (sacB s), CGA-N46 encoding sequence (cga-N46) and stop codon were tandem constructed into the muticloning sites of pSBPTQ. The engineered expression plasmids which bore monocistronic, bicistronic and tricistronic expression cassettes of the CGA-N46 gene were transformed into the competent B. subtilis strain DB1342. The expression results of the engineered strains DB1342(p-N46), DB1342(p-2N46) and DB1342(p-3N46) demonstrated that monocistronic and bicistronic expression systems produced low amounts of CGA-N46 while the tricistronic expression system improved the production of the peptide. Our results are helpful for improving the production of small peptides by engineered strains.


INTRODUCTION
Nosocomial bloodstream fungal infection remains a serious problem amongst hospitalized patients.Candidaemia is reported to be a major cause of morbidity and mortality (Ortega et al., 2011).The mortality of invasive Candida is unacceptably high and approaches 40% (Wisplinghoff et al., 2004;Gudlaugsson et al., 2003;Diekema et al., 2003).Azoles drugs are commonly used to treat infections caused by C. albican.However, there has been considerable evidence, which has proved that these medicines are becoming ineffective.The reasons for this are the appearance of drug resistance of candida strains and side effects of azoles in some individuals.On the contrary, peptide resistance is uncommon (Foubister, 2003).
Chromogranin A (CGA) is a ubiquitous secreted protein by most endocrine cells and it is present at nanomolar concentration in the human vascular system.In recent studies, some properties of the CGA N-domain have been described.Vasostatin-I (CGA1-76), which is the natural fragment of chromogranin A, is able to kill a large variety of fungi and yeast cells in micromolar range (Lugardon et al., 2000).CGA-N46, which is a derived peptide containing the 31st to 76th amino acid of the Nterminus of CGA, has special antagonistic activities to C. albican (Li et al., 2006).However, the mechanism has not been established yet, which hampers its application in drug exploration.With a study about the CGA-N terminus fragment, Lugardon and colleagues proposed that the destabilization of fungal cell wall and plasma membrane, together with intracellular inhibition of calmodulindependent enzymes, might be the reason by which vasostatin-I and chromofungin could inhibit the growth of fungi (Lugardon et al., 2001).Another related study suggested that vasostatin-I and CGA47-66 at concentrations of 5-10 nmol/l might engage in electrostatic as well as hydrophobic interactions with membrane-relevant phospholipids at physiological conditions.In particular, vasostatin-I enhanced the fluidity of saturated species of phosphatidylserine (Blois et al., 2006).Both of these mechanisms differ distinctly from each other.Therefore, these conclusions appear not to be solid and may need to be investigated further.To gain a deeper understanding of the antifungal mechanism of CGA-N46 and the need of drug development for future clinical application, a wealthy availability of CGA-N46 appears to be necessary.In addition, gene engineering is needed for its mass production.
Gene engineering approach is an effective means for large-scale production of peptides in vitro.In the last decade, many small peptides have been produced successfully using recombinant DNA technology.The most commonly used host cell is E. coli because of its fast growth and well-established expression system.However, expression of small peptides using E. coli as the host cell has been hampered by some barriers: (1) the formation of inclusion makes the isolation and purification of peptides difficult; (2) the susceptibility to proteolytic degradation (Zhong et al., 2006).
Fusion expression could overcome proteolytic degradation at certain level; however, it needs expensive and special cleavage enzyme to get the target protein.Construction of expression plasmids to express tandem repeat peptides is another approach to solve the above problems (Lee et al., 2002;Lennick et al., 1987) but products' multimerization may affect their native properties.
The polycistronic expression method is usually used to express a protein complex (Tan et al., 2005;Hierro et al., 2005;Shim et al., 2012).Construction of a polycistronic expression plasmid to express multiple copies of the expression cassette of one single exogenous gene in one plasmid may be one of the effective approaches to overcome the above barriers.Here, the expression of one single gene, namely cga-N46, via a polycistronic strategy in order to increase its expression level is described.

Plasmids and strains
The plasmid pSBPTQ,an E. coli-B.subtilisshuttle plasmid, was used to construct a recombinant expression plasmid in B. subtilis.It has the strong sacBpromoter of B. subtilis sacB p, which is a sucrose-inducible promoter.The pSC31-76 plasmid was used as the template of the CGA-N46 gene.Plasmid amplification, subcloning experiment and related gene manipulation were performed in E. coli DH5a.A four-protease-deficient B. subtilis strain DB1342 (his nprR2 nprE18 aprAS epr) was used for all the protein expression experiments.The plasmid pMD18-T was purchased from Takara Bio.Inc (Dalian, China).
There is a SacB signal peptide nucleotide sequence (sacB s) downstream of the SacB p in the pSBPTQ plasmid, a secreted expression vector of B. subtilis.There was no start codon (ATG) in primer P1.Primer P3 was designed according to the sequence of RBS of SacB.To make sure the stop of translation at desired position, the antisense stop codon TTA was added in primers P2 and P4.
To facilitate genetic manipulation and simplify the construction of polycistronic plasmids, pMD18-T was an intermediate vector for genetic cloning.There are XbaI (431 bp) and KpnI (450 bp) restriction sites in pMD18-T, but they are not at the flanks of the T cloning site (425 bp).To subclone the exogenous gene, which was cloned in the T site of pMD18-T, into pSBPTQ between XbaI and KpnI, a KpnI restriction site had to be added in the 5′ terminus of primer P4.

Construction of recombinant T plasmids
PCR products cga-N46, cga-N46-KpnI and sacBs-cga-N46, which had A-terminus, were recombined with pMD18-T plasmids, which had T-terminus, using T4 DNA ligase.The recombinant plasmids were transformed into E. coli DH5a and positive clones were screened by blue and white colony color.The target recombinant plasmids were identified by restriction endonuclease digestion.

Construction of polycistronic cga-N46 cassettes into pSBPTQ
Plasmid pSBPTQ had sacB promoter and signal peptide nucleotide sequence.The exogenous gene at the downstream of the signal peptide sequence of pSBPTQ could be expressed by B. subtilis.After digestion of the recombinant T plasmids and pSBPTQ with KpnI and XbaI, these two fragments were linked together by T4 DNA ligase.The positive recombinant plasmids were digested to verify the presence of cistronic cassettes.The target recombinant plasmids were transformed into B. subtilis DB1342 competent cells for protein expression.B. subtilis DB1342 competent cells were prepared by Spizizen method (Spizizen, 1958) and positive colonies could grow in LB agar containing 10 μg/ml kanamycin.

Expression analysis of CGA-N46
The seed culture was prepared by transferring a loop of engineered B. subtilis DB1342 cells from a fresh culture grown on LB agar into 25 ml LB broth (10 μg/ml kanamycin) in a 250 ml Erlenmeyer flask.The flask was incubated in a shaker (230 rpm) at 37°C for 16 h.
The expression process was carried out in Erlenmeyer flasks (250 ml) containing 50 ml aliquots of 2×MSR (modified super rich medium, 5% w/v yeast extract, 3% w/v peptone, 0.6% w/v K2HPO4 and 0.6% w/v glucose) supplemented with 10 μg/ml kanamycin.The flasks were inoculated with the seed culture at 5% v/v and incubated in a shaking incubator (230 rpm).Protein expression was induced after culturing for 3 h by the addition of 2% sucrose.Culture samples were collected at 30 h after induction and were

Assay of antifungal activity
Antifungal activity of the filtered sucrose-induced culture broth of B. subtilis DB1342(p-3N46) was examined by well diffusion assays.One hundred microliter of C. albican suspension (approximately 10 5 cfu/ml) was spread onto potato dextrose agar plates.The plates were then aseptically punched with 6-mm holes in the agar using a cork borer and 100 μl 0.22μm-filtered culture broth of B. subtilis DB1342 (p-3N46) was added to the wells.For each tested plate, one well was filled with the filtered culture broth of B. subtilis DB1342(pSBPTQ) and the filtered sucrose-induced culture broth of B. subtilis DB1342(p-N46) served as control.The plates were left undisturbed to allow the solvent to diffuse into the agar and then incubated at 28°C overnight.Another 100 μl of filtered culture broth was added until the total culture time was 36 h.Diameters of growth inhibition zone were measured in millimeters (Aslam et al., 2011).The experiment was carried out in triplicate and the average value was recorded.

Construction and screening of recombinant plasmid pT -N46-KpnI
PCR fragments of cga-N46-KpnI were recombined with pMD18-T plasmids.The target recombinant plasmids were screened by restriction endonuclease KpnI digestion.Because there was one KpnI digestion site on pMD18-T and a second one was introduced via cga-N46-KpnI, two KpnI digestion sites were present on the recombinant plasmids.The KpnI on pMD18-T was at 450 bp site, the T cloning site was at 425 bp of pMD18-T and the fragment length of cga-N46-KpnI was 164 bp.Therefore, when the recombinant plasmids from cga-N46-KpnI and pMD18-T were digested by KpnI, there were two possible results for the fragment length of the digested recombinant plasmids: 25 bp or 189 bp due to the insert orientation of the fragment cga-N46-KpnI.The plasmids with 189 bp fragment were the target recombinant plasmids (Figure 1) and named as pT-N46-KpnI.

Construction and screening of recombinant plasmid pT-N46
PCR fragments of cga-N46 were recombined with pMD18-T plasmids.To test whether the fragment cga-N46 was inserted into pMD18-T, the recombinant plasmids were screened by double restriction digestion using NdeI and NheI endonucleases.Results showed that there were two kinds of fragments digested out of the recombinant plasmids because of the orientation of the inserted fragment cga-N46.Because the T cloning site was at 425 bp of pMD18-T, the lengths of the fragments digested out of recombinant plasmids were calculated to be 240 bp and 398 bp.The recombinant plasmids out of which the length of a 240 bp fragment could be digested were the target plasmids (Figure 2) and named as pT-N46.

Construction and screening of recombinant plasmid pT-SN46
Fragments of sacBs-cga-N46 were recombined with pMD18-T plasmids.In order to check if the fragment of sacBs-cga-N46 was inserted into pMD18-T and screen the target recombinant plasmids, double restriction endonucleases NdeI and NheI digestion were used.There were two kinds of fragments digested out of the recombinant plasmids because of the orientation of the inserted fragment sacBs-cga-N46.The fragment lengths digested out of recombinant plasmids were calculated to be 240 bp and 540 bp.The recombinant plasmids out of which could be digested out a 240 bp fragment were the target plasmids (Figure 3) and named as pT-SN46.

Construction and screening of recombinant plasmid pT-2N46
To construct the recombinant plasmid containing double copies of cga-N46, plasmids pT-N46 and pT-SN46 were digested by NdeI and NheI respectively.Fragments containing cga-N46 from pT-N46 and pT-SN46 were linked by T4 DNA ligase.The protocol of pT-2N46 construction is shown in Figure 4.

Construction and screening of recombinant plasmid pT-3N46
Plasmid pT-2N46 was digested by NdeI and NheI and the fragment containing cga-N46 was recirculated.Plasmid pT-2N46 was also digested by NdeI and SpeI and the fragment containing cga-N46 was recirculated.Two recirculated fragments were linked together by T4 DNA ligase.The positive plasmids were named as pT-3N46.The progress of construction is shown in Figure 5.

Construction of recombinant pSBPTQ plasmids
There was a MCS including KpnI and XbaI downstream of the promoter and signal peptide of sacB in pSBPTQ.The fragments digested from pT-N46, pT-2N46 and pT-3N46 with KpnI and XbaI were linked with the fragments digested from pSBPTQ with the same two endonucleases by T4 DNA ligase.The recombinant plasmids containing monocistronic, bicistronic and tricistronic cassettes of cga-N46 were constructed (Figure 6).Each cistron could express one copy of CGA-N46.

Expression of monocistronic, bicistronic and tricistronic cassettes of cga-N46
To gain the soluble secreted peptide CGA-N46, the engineered B. subtilis DB1342 cells, which bored recombinant plasmids p-N46, p-2N46, and p-3N46 respectively, were cultured in 2×MSR medium containing 10 μg/ml kanamycin for 3 h.CGA-N46 gene expression was then induced by the addition of 2% sucrose.Culture broth of each engineered strain was collected after 30 h.The yields of CGA-N46 in culture broth were compared by 16.5% SDS-PAGE (Figure 7).
The results show that CGA-N46 was mainly expressed in DB1342 (p-3N46) and secreted into culture broth whereas a very limited amount of CGA-N46 was expressed in DB1342(p-N46) and DB1342(p-2N46).

Antifungal activity
The antifungal activity of the filtered sucrose-induced culture broth of B. subtilis DB1342(p-3N46) was tested.The average diameter of the clear zone in the well diffusion assay was about 20 mm while there was nearly  no clear zone for B. subtilis DB1342(pSBPTQ) culture broth and a little clear zone of culture broth of B. subtilis DB1342(p-N46) (Figure 8).

DISCUSSION
In this study, we expressed the cga-N46 gene by a tricistronic expression method in B. subtilis to improve the production of CGA-N46.
The tricistronic expression plasmid was constructed by Yang and colleagues (2006) using conventional plasmid construction methods to express Echistatin.Three pairs of primers, six kinds of restriction endonucleases, three times of PCR amplification, three times of endonuclease digestion and ligation reactions were needed in the conventional plasmid construction methods.To overcome the shortage of the conventional construction methods, ) via digestion and ligation as the above strategy.Finally, DNA fragments with multiple exogenous genes were subcloned into the expression plasmid at downstream of the promoter through the corresponding restriction digestion.Thus, the recombinant plasmids bearing a polycistronic expression cassette could be obtained.
Compared with the conventional plasmid construction technology, the construction method of polycistronic ex- pression plasmid established in the present study has the following advantages: (1) Fewer kinds of restriction endonucleases are needed.2n (n stands for the number of cistrons) kinds of endonucleases are needed in the conventional plasmid construction technology.In contrast, no matter how many cistrons, only five kinds of endonucleases are needed in this method.(2) Fewer primers are needed.n (n stands for the number of cistrons) pairs of primer are needed in the conventional plasmid construc-tion technology.On the contrary, only two pairs of primers are required regardless of how many cistrons are going to be recombined.(3) Fewer experimental steps.For example, if we construct a nine-cistronic expression plasmid using the conventional plasmid construction technology, 9 PCR reactions, 9 digestion and ligation reactions will be needed.However, only 2 PCR reactions, 5 digestion and ligation reactions are required by the method reported in this study.(4) The number of cistrons is unrestricted.The number of cistrons is limited by the number of restriction sites in the MCS of the expression plasmid in the conventional plasmid construction technology, whereas the number of cistrons is unrestricted in this method.Furthermore, this polycistronic plasmid construction method is simple and widely applicable.This method can be used to express all proteins, especially small peptides.On the other hand, the use of an intermediate plasmid in this method is not necessary.We can directly manipulate the exogenous gene in the expression plasmid and the resultant recombinant plasmids can be directly transformed into host cells, including both prokaryotic and eukaryotic cells.
Using the simple and efficient method established in this study, three kinds of polycistronic expression plasmids, namely p-N46, p-2N46 and p-3N46, were constructed.CGA-N46 was expressed and secreted from three engineered strains, namely DB1342(p-N46), DB1342(p-2N46) and DB1342(p-3N46).The results presented in Figure 7 demonstrated that the tricistronic expression cassette could express more target peptide than the monocistronic and bicistronic expression cassettes.
B. subtilis is not a pathogenic microbe and therefore it is harmless to human beings and livestock.B. subtilis also has a well-secretion system (Wong, 1995;Wu and Wong, 1999).Thus, it is an effective expression host.This is the reason why B. subtilis was used as expression host in this study.The expressing product was secreted into the medium to facilitate purification.
As expected, we increased the production and realized secreted expression of the antifungal peptide CGA-N46 by polycistronic expression in B. subtilis in this study.However, the yield of CGA-N46 in B. subtilis is still a practical limitation.Further research will be devoted to remove this limitation or chose another expression host.Nevertheless, we believe that the method established in this investigation may have wider application in such areas of protein expression in the future.

Figure 1 .
Figure 1.Construction and screening of the recombinant plasmid pT-N46-KpnI.Plasmid pSC31-76 was amplified using the oligonucleotide primers P1 and P4.The PCR fragment cga-N46-KpnI was recombined with the pMD18-T plasmid using T4 DNA ligase.There were two KpnI sites on the recombinant plasmids.One was at 450 bp site of pMD18-T and another one was at 3′ terminus of cga-N46-KpnI, which was inserted in the T cloning site at 425 bp of pMD18-T.The fragment length of cga-N46-KpnI was 164 bp.When the recombinant plasmids were digested by KpnI, there were two possible results for the length of the fragments digested out of recombined plasmids: 25 bp and 189 bp due to the insert orientation of the cga-N46-KpnI fragment.

Figure 2 .
Figure 2. Construction and screening of the recombinant plasmid pT-N46.Plasmid pSC31-76 was amplified using the oligonucleotide primers P1 and P2.The PCR fragment cga-N46 was recombined with the pMD18-T plasmid using T4 DNA ligase.There were one NdeI site and one NheI site on recombinant plasmids.The NdeI was at 185 bp site of pMD18-T, and NheI was at the 3′ terminus of cga-N46 which had been inserted in the T cloning site at 425 bp of pMD18-T.The fragment length of cga-N46 was 158 bp.When the recombinant plasmids were digested by NdeI and NheI, there were two possible results for the length of the fragments digested out of the recombined plasmids: 240 bp and 398 bp due to the insert orientation of the cga-N46 fragment.

Figure 3 .
Figure 3. Construction and screening of the recombinant plasmid pT-SN46.Plasmid pSC31-76 was amplified using the oligonucleotide primers P3 and P4.The PCR product sacBs-cga-N46 was recombined with the pMD18-T plasmid using T4 DNA ligase.There were one NdeI site and one NheI site on the recombinant plasmids.The NdeI was at 185 bp site of pMD18-T and NheI was at the 3′ terminus of the sacBs-cga-N46 fragment, which had been inserted in the T cloning site at 425 bp of pMD18-T.The fragment length of sacBs-cga-N46 was 300 bp.When the recombinant plasmids were digested by NdeI and NheI, there were two possible results for the length of the fragment digested out of the recombined plasmids: 240 bp and 540 bp due to the insert orientation of the sacBs-cga-N46 fragment.

Figure 4 .
Figure 4. Construction of the recombinant plasmid pT-2N46.Plasmids pT-N46 and pT-SN46 were digested by NdeI and NheI respectively.The fragment from pT-N46 containing cga-N46 and the fragment from pT-SN46 containing sacBs-cga-N46 were recirculated and linked by T4 DNA ligase.

Figure 5 .
Figure 5. Construction of the recombinant plasmid pT-3N46.Plasmid pT-2N46 was digested by NdeI and NheI and recirculated the sacBs-cga-N46 fragment.Plasmid pT-2N46 was digested by NdeI and SpeI, and recirculated the cga-N46 fragment.Two recirculated fragments were linked together by T4 DNA ligase.

Figure 6 .
Figure 6.Construction of the recombinant pSBPTQ plasmids.Fragments from pT-N46, pT-2N46 and pT-3N46 digested with KpnI and XbaI were linked with lining plasmid pSBPTQ digested with the same two endonucleases.The recombinant plasmids p-N46, p-2N46 and p-3N46 were constructed.

Figure 8 .
Figure 8. Antifungal activity of the filtered sucrose-induced culture broth of B. subtilis DB1342(p-3N46).The culture broth was filtered by 0.22 μm filter paper.One hundred μl of the filtered culture broth was added into the hole of the plate.After the plates were incubated overnight, another 100 μl filtered culture broth was added into the hole.After incubating for a total of 36 h, the diameters of the clear zone were measured in millimeters.A. Culture broth of B. subtilis DB1342(pSBPTQ).B. Culture broth of B. subtilis DB1342(p-3N46).C. Culture broth of B. subtilis DB1342(p-N46).