Aiming to achieve a fast and economical way to evaluate the solubility of RP, we selected two commonly used expression vectors pQE-80L (Qiagen) and pET-32a (Novagen) as the starter plasmids for the suite generation thus giving rise to T5 or T7 based vectors. In order to provide a parallel cloning of the target gene and an easy protein purification method, all the generated vectors contain the same insertion site and antibiotic resistance (ampicillin), an N-terminus His-Tag with the tobacco etch virus (TEV) recognition site and a C-terminus strep-Tag II (Figure 1; Table 1). In addition, we introduced several solubility enhancing proteins including MBP, Trx, DsbC, SUMO, and CelDnc, in combination with the two promoters (T5 or T7). An extra serine residue was added after the TEV site to decrease steric effects and improve cleavage. This can be avoided by not including it in the forward primer. This extra codon also generates a BamHI site at the beginning of the gene so it can be useful for analysis of clones or to do a restriction based method if preferred (Figure 1).

In order to evaluate the expression capabilities and functionality of this new vector suite we selected green fluorescent protein (GFP) as control protein and two “difficult to express” RP such as DprE1 and the MAP kinase 4 from Leishmania major (MPK4). All of them were cloned into 12 different vectors and their expression was evaluated. The results showed that all the GFP constructs were produced soluble and at the expected molecular weight. Fractions treated with TEV showed the correct cleavage and release of GFP protein and fusion partner (Figure 2A). The construct DsbC-GFP under the control of T7 promoter was the less productive when working at 37°C. This was over-passed when the expression was done at 17°C over night (ON) where an increment of cleaved proteins was obtained in most of the cases (Figure 2A).

For the case of DprE1 constructs, we can see that despite a correct growth and induction conditions in the culture, it was not possible to obtain any expression of this RP when fused only to a Histag. In contrast, fusion of DprE1 with MBP, Sumo, Trx, and CelDnc give a good soluble production and only low yields account for the DsbC/DprE1 construct (Figure 2B; Table 2). Also, there was an effect of the induction temperature and promoter strength in protein expression where DprE1 was expressed with higher yields at 37°C compared to 17°C and with the T5 promoter compared with T7 for most of the cases. Interestingly, our results suggested that DprE1 fused with CelDnc (in the condition T5-37°C) appear to be one of the most overexpressed fused proteins. For the case of DprE1/CelDnc in T7 at 17°C, there was no cell growth. Finally, the treatment with TEV revealed that DprE1 RP fused with all these enhancers remains in a soluble state confirming an important improvement expression after usage of this vector suite (Figure 2B).

Concerning MPK4 our results showed that of the 12 constructs only 2 gave a band at the expected molecular weight. These correspond to the construct pT7-DsbC-MPK4 and pT7-MBP-MPK4 (Figure 2C). In both cases TEV protease was able to cleave the fusion but only in the pT7-DsbC-MPK4 constructs it was possible to get a soluble protein after cleavage (Figure 2C, TEV treatment section). In order to confirm this result and validate our suite vector we proceed to perform a large scale purification with this construct. Our results showed that after protein purification by IMAC it is possible to obtain the DsbC-MPK4 fusion in a soluble manner and with a yield of 6 mg/l (Figure 2D). Oligomeric state analysis of the DsbC-MPK4 fusion, revealed that the eluted peak is maintained as a soluble decameric oligomer with an apparent molecular weight of around 650 kDa (Figure 2D). This result was verified by dynamic light scattering (data not shown). Despite the fact, a great part of the MPK4 protein precipitate after TEV treatment, an interesting and scalable amount of this protein remains in a soluble form (Figure 2D).

Altogether these results, underline the importance of this new vector suite as an improved tool for the soluble expression of DprE1 and MPK4 proteins and suggest that it can be very valuable for the expression of other “difficult to express” RP.

After expressing the new construct (CelDnc), we found out that it is expressed at high yields (>400 mg/l) in a soluble monomeric and functional form which in turn maintains thermostable characteristics as the entire version (Figures 3A,B). So, we wondered if this extreme solubility and stability could help in the production and folding of other target proteins. In this regard, we fused CelDnc to the N-terminus of the protein DprE1. The results showed that the fusion was successfully produced in a soluble manner and that after TEV treatment and gel filtration purification it remains soluble, monomeric and it was able to retain a FAD binding property as expected for this protein (peaks at 360 and 460 nm; Figure 3C). The final yield was of 7 mg/l which corresponds to more than 17 times improvement in soluble protein expression when compared with no fusion (<0.4 mg/l). Moreover, the same experiment done with MBP fusion resulted in a final yield for DprE1 of 2.8 mg/l (data not shown), demonstrating the usefulness of CelDnc as a solubility enhancer of RP.

These results suggest that the construct CelDnc is an interesting new solubility enhancer that could be taken into account for the expression screening of “difficult to express” RP.

For the generation of the vector suite we used a modified version of the pQE80L (Qiagen) as the starter plasmid, that contained a TEV cleavage site after the Histag separated by a GSGS linker (pQE80L-TEV). In a first step we cloned the gene DprE1 into this vector and added the different modules for the vector suite (linkers, strepTag and different fusion proteins) thus generating the T5 series. Then the entire constructs were cloned into the vector pET32a in order to generate the T7 series.

All PCR were done using Phusion polymerase (Finnzymes). For the amplification of the fragments (megaprimer generation) conditions were 30 s at 98°C and 28 cycles of 98°C for 10 s, 59°C for 1 min and 72°C for 1 min with a final extension step at 72°C for 5 min and PCR products were purified by agarose gel. The generated megaprimers contained 30 bp in both ends that overlaps with the insertion site in the destination vectors. The integration into the vectors was done by RF cloning (Unger et al., 2010) and the RF reaction was as follows: 30 s at 98°C and 30 cycles of 98°C for 10 s, 60°C for 1 min and 72°C for 5 min with a final extension step at 72°C for 7 min. For RF reactions 120 ng of megaprimers and 30 ng destination vector were used. 20 μl were digested with 2 μl Fast Digest DpnI (Thermo) for 15 min at 37°C in order to remove parental plasmid, and 5 μl were used to transform 50 μl of competent DH5α E. coli cells. Positive clones were confirmed by colony PCR by using Taq polymerase (Invitrogen) with the same primers used for megaprimer generation. Colony PCR was as follows, 95°C for 3 min, 25 cycles of 95°C for 30 s, 60°C for 30 s and 72°C for 2 min followed by a final extension step at 72°C for 5 min. Positive colonies were selected for plasmid extraction and confirmed by sequencing.

The gene for DprE1 was amplified from M. smegmatis genomic DNA using the primers QE3790For and QE3790Rev for the generation of the megaprimer (Table 1). The product was cloned into the vector pQE80L-TEV by RF cloning to generate the construct pDprE1. The genes coding for CelDwt or the truncated version CelDnc (residues 32–577), were amplified from the plasmid pCT603 (Chaffotte et al., 1992) with the primers CelDwtNFor and CelDwtCRev for CelDwt and primers CelDtruncNFor and CelDtruncCRev for CelDnc (Table 1) and cloned by RF in the same vector to generate the constructs pCelD and pCelDnc. The construct pDprE1 was used for the insertion of CelDnc in the 5′ of DprE1 (between the HisTag and the GSGS linker, Figure 1). CelDnc was amplified from the pCelDnc construct using primers CelDInsFor and CelDInsRev. The forward primer was designed also to add a GSSG linker to separate the HisTag from the fusion partner generating the construct pCelD-DprE1. The generated constructs (pDprE1 and pCelD-DprE1) were then used to add the last module of the vector, the C-terminal strepTag II. The strepTag II was inserted at the C-terminus separated by a GSGS linker with primers strepCterFor and strepCterRev (Table 1) for the generation of the vector pT5-DprE1 (HisTag alone) and pT5-CelD-DprE1 (CelDnc fusion). The primers anneal each other, so they were used without addition of DNA for the generation of the megaprimer. The generated pT5-CelD-DprE1 vector was then used for the insertion and replacement of CelDnc by other fusion partners. In this regard the primers SumoFor and SumoRev; TrxFor, and TrxRev; MBPFor and MBPRev and DsbCFor and DsbCRev were used for the insertion of Sumo, TrxA, MBP, and DsbC, respectively, (Table 1). The genes were amplified from Saccharomyces cerevisiae for Sumo, pET32a (Novagen) for TrxA, pMAL (New England Biolabs) for MBP; and E. coli genome for DsbC. By this way, the T5 vector series was completed. All 6 vectors were confirmed by sequencing with the QEFor and QERev plasmid primers. For the case of MBP and CelDnc constructs internal primers were also used in order to cover the entire sequence. The last step was to transfer the modules into a T7 context. To do this, we selected the pET32a (Novagen) as a destination vectoramplifying the entire cassette from T5 series (from MRGS-HisTag up to the strepTag II for the different fusions) with the primers T5T7For and T5T7Rev and replacing the expression cassette of the pET32a vector. The generated megaprimers were used for the RF reactions. By this way the vector suite was completed containing the gene DprE1 in all 12 vectors for expression screening.

Leishmania major MPK4 gene was amplified with primers MPK4For and MPK4Rev from a pGem vector containing the gene. GFP was amplified with primers GFPFor and GFPRev from a pET vector containing a GFP variant that is well expressed in E. coli (Waldo et al., 1999).

The 12 vectors were added to 12 different PCR tubes, and the amplified products were used as megaprimers for the RF reaction using the HF buffer from Phusion polymerase. After digestion of 20 μl PCR products with 2 μl DpnI, chemical competent cells were transformed with 5 μl RF reaction in a PCR machine with the following program: 30 min at 4°C, 45 s at 42°C, 3 min at 4°C, addition of 100 μl of LB, 1 h at 37°C, and plating of 100 μl in agar plates containing ampicillin. Four colonies for each construct were selected and confirmed by colony PCR and sequenced. After the analysis we found out that in most cases all were positive (or at least three of four were positive) giving a percentage of success of more than 80%.

Chemocompetent Rosetta-pLysS cells were transformed with 5 μl of purified plasmids as described above and then incubated in a shaker ON at 37°C in 1 ml of LB with chloramphenicol and ampicillin in a 96 × deep-well plate. 100 μl of ON culture were used to inoculate 4 ml of Terrific Broth in 24 × deep-well plates by duplicate. Cultures were incubated at 37°C until D.O.₆₀₀ reached 1.0–1.2. At that moment one plate was induced with 1 mM IPTG and left at 37°C for 4 h. The other 24 deep-well was incubated at 17°C for 15 min to cooling it and then induced with 1 mM IPTG ON at the same temperature. After induction time was reached, cells were harvested, resuspended in 1 ml lysis buffer (50 mM Tris pH 8.0; 300 mM NaCl, 10 mM imidazol, 0.5 mg/ml lysozyme) and frozen at -80°C. After thawing cells, 10 units of DNase I and 10 μl of 2M MgSO₄ were added and incubated with shaking for 20 min at 20°C. Then 200 μl of Nickel beads (Qiagen) equilibrated in binding buffer (50 mM Tris pH 8.0; 300 mM NaCl, 10 mM imidazol) were added to cell extracts and incubated for 15 min at 20°C. Cell extracts were then transferred to a 96×-well filter plate assembled in a vacuum device, and bound protein was washed with 2 ml of binding buffer. An additional wash step was done with 2 ml of binding buffer containing 50 mM imidazol. Elution was done with 160 μl of elution buffer [50 mM Tris pH 8.0; 300 mM NaCl, 500 mM imidazol; for a detailed protocol, see (Saez and Vincentelli, 2013)]. Eluates were divided in two groups for evaluation of uncleaved protein and assessment of TEV cleavage ON at 18°C. Samples were then loaded into an E-PAGE 96 acrylamide gel (Invitrogen).

Expression screening and purification of MPK4 constructs was made in a similar way than for GFP and DprE1 but only 17°C of induction was evaluated. Purification steps were the same but the pipeting scheme was done automatically by using a TECAN Freedom EVO®200. Expression analysis was done also automatically by using a Labchip GX II (Caliper, USA) microfluidic detection system.

DsbC-MPK4 was expressed in Terrific Broth (TB) supplemented with ampicillin and chloramphenicol and induction was done at D.O₆₀₀: 1.2 ON at 17°C with 1 mM IPTG. Pellets were resuspended in lysis buffer and frozen at -80°C. After thawing, the pellets were sonicated and centrifugated at 15.000 × g. Soluble fraction was injected in a 1 ml IMAC column (GE Healthcare) equilibrated in binding buffer. Elution was done in a linear gradient of 5–100% B in 10 column volumes (CV) with elution buffer. Purified protein was cleaved with TEV protease in a 1:30 protein:enzyme ratio and dialyzed against cleavage buffer (50 mM Tris pH 8.0; 150 mM NaCl, 1 mM DTT) ON at 8°C. Sample was filtered through 0.22 μm to remove precipitates, and analyzed by SDS-PAGE.

Production of CelD and CelDnc was done in M15pREP4 from the constructs pCelD and pCelDnc, respectively, in 1 l 2YT supplemented with ampicillin and kanamycin, and induced with 1 mM IPTG at D.O. 1.0 ON at 37°C. IMAC was done like for the case of DsbC-MPK4 but using a 5 ml column and only half of the soluble fraction was used. TEV cleavage was done as before and desalted in order to remove imidazole. The reaction was injected in a second IMAC under same conditions as above and the flow through containing the cleaved protein was injected in a Superdex 200 16/60 (GE Healthcare) equilibrated with buffer 40 mM Tris pH 7.7.

Differential scanning calorimetry (DSC) experiments were carried out in PBS, in a VP-DSC instrument (Microcal, Northampton, MA, USA) and data analyzed with the software supplied with the equipment. The temperature was increased at 1°C per minute from 30 to 80°C, and proteins were added at concentration of 1 mg/ml for CelD and CelDnc.

Induction of p5DprE1, p5CelDnc-DprE1 and p5MBP-DprE1 were done in M15pREP4 with 1 mM IPTG in 1 l 2YT supplemented with ampicillin (100 μg/ml), kanamycin (50 μg/ml) and 15 μM FAD at D.O.: 1.0–1.2 during 4 h at 37°C. Cells were harvested, resuspended in lysis buffer and frozen at -80°C. After thawing the cells, were lysed and protein purified as before. Purified protein was cleaved with TEV in a 1:30 ratio, and dialysed against cleavage buffer. The product was then purified by a second IMAC and injected in a Superdex 200 16/60 equilibrated with buffer 25 mM Tris pH 8.0; 150 mM NaCl.