Karine Rottier

Alexandre Faille

Thomas Prudhomme

Cécile Leblanc

Christian Chalut

Stéphanie Cabantous

Christophe Guilhot

Lionel Mourey

Jean-Denis Pedelacq
email: denis.pedelacq@ipbs.fr

Detection of soluble co-factor dependent protein expression in vivo : Application to the 4'-phosphopantetheinyl transferase PptT from Mycobacterium tuberculosis

Keywords: Split-GFP assay, domain trapping, 4'-phosphopantetheinyl transferase, polyketide, tuberculosis Acyl carrier protein synthase AnTet: Anhydrotetracycline BSA: Bovine serum albumin CoA: Co-enzyme Differential scanning fluorimetry Green fluorescent protein IMAC: Immobilized metal ion affinity chromatography IPTG: Isopropyl β-D-1-thiogalactopyranoside

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Introduction

High-level expression of a protein target in a soluble form is a prerequisite to its downstream functional and structural characterization. Escherichia coli, the most widelyused heterologous host for the production of recombinant protein, has incommensurable benefits over existing systems: well-characterized genetics, easy to transform, easy growth, and inexpensive. The diversity of E. coli strains and expression vectors publicly available now permits the production of most cytoplasmic proteins. Engineered E. coli strains resistant to the toxicity of membrane protein expression have been described 1; 2; 3 . Good progress has also been made in overexpressing eukaryotic proteins [START_REF] Ito | Using codon optimization, chaperone co-expression, and rational mutagenesis for production and NMR assignments of human eIF2 alpha[END_REF] , a major breakthrough considering the lack of post-translational machinery and the limiting codon usage in the E. coli host. Sometimes, proteins partition almost exclusively into "inclusion bodies" when overexpressed in heterologous hosts as a result of misfolding, aggregation, and intracellular accumulation. To circumvent this issue, cell-free protein synthesis systems and refolding strategies have been implemented [START_REF] Chow | The REFOLD database: a tool for the optimization of protein expression and refolding[END_REF] .

As an alternative to the time-consuming and rate-limiting steps that best characterize the search for optimized expression and solubility conditions in heterologous hosts, new strategies have emerged that specifically focus on modifying the underlying DNA sequence. With the advent of the error-prone PCR technique [START_REF] Mokbel | Telomerase activity and lymphovascular invasion in breast cancer[END_REF] , introducing mutations that enhance solubility and stability of a protein of interest while retaining its enzymatic activity has now become possible 7; 8; 9 . Technology improvements have also considerably reduced the cost of high-throughput screening of DNA fragment libraries thanks to the development of colony picking, arraying and liquid handling robots [START_REF] Yumerefendi | ESPRIT: an automated, library-based method for mapping and soluble expression of protein domains from challenging targets[END_REF] coupled with biotechnology tools. Strategies to filter out DNA fragments that do not encode authentic regions or domains of a protein of interest have been implemented 11; 12 .

In this paper, we detail a new application of the split-GFP complementation assay [START_REF] Cabantous | Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein[END_REF] as a diagnostic tool to investigate the solubility of co-factor dependent proteins expressed in vivo. We used this system onto the 4'-phosphopantetheinyl transferase PptT from Mycobacterium tuberculosis, a coenzyme A (CoA) binding enzyme essential for the survival of mycobacteria 14; 15; 16 . E. coli assays indicated that the full-length enzyme was the most soluble among all the variants tested. We have shown that the natural endogenous CoA and divalent metal ion Mg 2+ , which play a crucial role for PptT to carry out its function, are also a prerequisite for its stability overtime. This easy-to-use split-GFP system can be generalized to detect soluble co-factor dependent protein expression in vivo whenever the presence of cofactors is required for folding and stability.

Materials and Methods

PptT small scale expression and solubility tests. The pptT gene from M. tuberculosis was amplified from genomic DNA using top and bottom primers listed in supplementary table 1. The NdeI/BamHI digested pptT was then inserted into commercially available pET28 vectors (Novagen, Madison, WI, USA) with no 6His tag, N-terminal or Cterminal 6His tag. pET vectors allowing the production of PptT with MBP [START_REF] Leblanc | 4'-Phosphopantetheinyl Transferase PptT, a New Drug Target Required for Mycobacterium tuberculosis Growth and Persistence In Vivo[END_REF] or SUMO (Invitrogen, Carlsbad, CA, USA) fused to its N-terminus were also tested. Ligated plasmids were transformed into chemically competent E. coli BL21(DE3) cells (Invitrogen, Carlsbad, CA, USA). Transformed cells were plated onto Luria-Bertani (LB) agar plates containing 35 mg/ml kanamycin, allowing them to grow overnight at 32ºC. The resulting clones were grown at 37ºC in 1ml cultures using 35 mg/ml kananycin. Cells were induced in exponential phase with 1mM IPTG for 3 h. Cell culture pellets of 1 ml of each fragment were separately resuspended in 40 ml TNG buffer (150 mM NaCl, 100 mM Tris-HCl pH=7.5, 10% (v/v) glycerol) and sonicated. The lysate was fractionated by centrifugation to yield the soluble and pellet fractions. The pellet fraction was washed twice with 100 ml TNG buffer, centrifuged and resuspended in the same starting volume.

Samples corresponding to the soluble (S) and pellet (P) fractions were resolved on a 4-20% gradient Criterion SDS-PAGE gel (Bio-Rad, Hercules, CA, USA). Protein samples were stained using Gel Code Blue stain reagent (Pierce, Rockford, IL) and imaged using a GS-800 Calibrated Densitometer (Biorad, Hercules, CA, USA).

Cloning and in vivo split-GFP solubility screen.

Full-length and truncated variants of the M. tuberculosis PptT gene were amplified by PCR using a series of forward and reverse primers listed in supplementary table 1. Cleaned inserts from NdeI/SpeI restriction digests were ligated into the pTET ColE1 GFP11 vector and transformed into chemically competent BL21(DE3) cells containing the pET GFP 1-10 plasmid 13; 18 . Frozen cells were used to grow 1 ml Luria-Bertani (LB) medium in a 96-well deep-well plate containing 35 µg/ml kanamycin and 112 µg/ml spectinomycin until OD 600 reached ~1.0 at 37˚C. 50 µl of two successive 400-fold dilutions of the expressed constructs were plated onto a compartmentalized nitrovivose membrane and grew overnight at 32 C. The membrane was transferred to an LB/Agar plate containing the same antibiotics supplemented with 250 ng/ml anhydrotetracycline (AnTET) for 2 h at 32 C, and then moved back onto the original plate for 1 h at the same temperature. Finally, the membrane was transferred onto a plate with same antibiotics and 1 mM IPTG to induce the expression of the GFP 1-10 fragment at 32 C. Colonies were illuminated using an Illumatool Bright light System LT-9900 (http://www.lightools.com/) at excitation and emission wavelengths of 470 nm and 515 nm, respectively. Pictures were taken after 30 min complementation at 32 C.

In vitro split-GFP complementation assay. 5 ml LB-Spec cultures were grown at 37 C until OD 600 reached 0.5-0.7. After induction at 32 C with AnTET at a final concentration of 8 µg/ml and growth for an additional 4 h, cells were harvested by centrifugation at 20000g for 20 min. 20 l of PptT-GFP11 soluble fractions were mixed with 180 l of 0.35 mg ml refolded GFP1-10 in a 96 well microplate (Nunc-Immuno TM plate, Nunc, Rochester, NY), as previously described [START_REF] Cabantous | In vivo and in vitro protein solubility assays using split GFP[END_REF] . Fluorescence kinetics (λ exc =488 nm λ em =530 nm) were monitored with a FL600 Microplate Fluorescence Reader (Bio-Tek, Winooski, VT), at 3 min intervals, for 15 h. The background fluorescence of a blank sample (20 l E. coli lysate expressing irrelevant protein, 100 l of 0.5 mg/ml GFP1-10 and 100 l 0.5% BSA in TNG buffer) was subtracted from final fluorescence values. The blank was less than 30% the signal from the lowest target concentration (0.1 pmol SR-S11).

Full-length PptT production and purification.

Frozen cells from the full-length PptT constructs ligated into a pTet vector missing the GFP11 tag were used to start an overnight 3 ml LB-spectinomycin (112 µg/ml) culture at 32˚C prior to inoculation in baffled flasks containing 500 ml of the same media. Cells were allowed to grow for approximately 2 h at 37˚C before temperature was dropped from 37˚C to 20˚C. When OD 600 reached 0.5-0.7, cells were induced with AnTET at a final concentration of 8 μg/ml and grown for an additional 18 h prior to harvesting by centrifugation at 4000 g for 30 min and storage at -80°C. The pelleted cells were suspended in 40 ml of buffer A (100 mM Tris pH 8.0, 300 mM NaCl, 10 mM MgCl 2 , 50 µM CoA) and lysed by sonication (6 cycles of 30 s pulse, 50% amplitude, power 5) prior to centrifugation at 20,000 g for 1 h.

The supernatant was filtered (0.2 µm) and purified on an ÄKTA purifier system (GE Healthcare) following a two-step procedure combining an immobilized metal ion affinity chromatography (IMAC) and a size-exclusion chromatography (SEC). The lysate was loaded onto a 1 ml HisTrap HP (GE Healthcare) affinity column. The N-terminally His tagged PptT was eluted from the column with a step of buffer A supplemented with 120 mM imidazole. The eluted protein fractions were pooled and concentrated in Vivaspin®

Results

Expression of PptT in different bacterial vectors. We first cloned pptT into pET28 derived vectors prior to transformation into classical BL21(DE3) cells. Only in a situation

where the histidine tag is present at the N-terminal extremity of the protein that overexpression of PptT can be achieved. The construct is poorly soluble and expresses mainly as inclusion bodies after IPTG induction (Figure 1). To maximize the amount of soluble protein, one strategy was to screen conditions where the inclusion bodies could be refolded in a stable and active conformation. This was achieved in the presence of 100 mM MES at pH 5.5. Unfortunately, analytical gel filtration experiments indicated an aggregated form of the enzyme detected in the exclusion volume of the column and the activity of the enzyme could not be confirmed.

As an alternative, we cloned pptT into pET vectors with fusion partners MBP (Maltose binding protein) [START_REF] Baba Moussa | Discoupling the Ca(2+)activation from the pore-forming function of the bi-component Panton-Valentine leucocidin in human PMNs[END_REF] and SUMO (Small ubiquitin-related modifier) [START_REF] Mossessova | Ulp1-SUMO crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast[END_REF] , previously described as solubility enhancers of passenger proteins (see Materials and Methods). On the one hand, SUMO had no effect on the solubility of the fusion. On the other hand, the PptT-MBP fusion was mostly soluble and could be purified easily following a two-step chromatographic procedure using metal-affinity resin followed by size exclusion chromatography. Unfortunately, the enzyme precipitates massively after proteolytic cleavage of the C-terminal MBP tag, which indicates that the MBP fusion maintains PptT in a soluble form. This MBP-PptT fusion was used for the implementation of an enzymatic assay suitable for high-throughput screening of small molecule libraries [START_REF] Leblanc | 4'-Phosphopantetheinyl Transferase PptT, a New Drug Target Required for Mycobacterium tuberculosis Growth and Persistence In Vivo[END_REF] . as previously described [START_REF] Cabantous | In vivo and in vitro protein solubility assays using split GFP[END_REF] . Approximately one third of the enzyme could be expressed in a soluble form, a result that was further confirmed in vitro by quantification of the corresponding band intensities on a SDS-PAGE gel (data not shown). A two-step purification procedure combining immobilized metal ion affinity chromatography (IMAC) and size-exclusion chromatography (SEC) indicated that the protein was mostly aggregated and could precipitate within hours.

Split-GFP solubility screen of

Knowing the essentiality of CoA and Mg 2+ for PptT to carry out its function in vivo [START_REF] Quadri | Identification of a Mycobacterium tuberculosis gene cluster encoding the biosynthetic enzymes for assembly of the virulence-conferring siderophore mycobactin[END_REF] , we were wondering if these compounds could also help maintaining the enzyme in a stable conformation in vitro. Only in a situation where CoA and MgCl 2 are present in a large molar excess over PptT in the cell lysate and all along the two-step purification strategy that high yields of soluble recombinant protein can be attained. We then conducted a series of experiments using the Differential Scanning Fluorimetry (DSF) method [START_REF] Niesen | The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability[END_REF] to confirm the importance of the two protagonists onto the stability of PptT. We used Sypro Orange to detect the buried hydrophobic regions of the enzyme that become progressively exposed to solvent during a linear temperature ramp from 20˚C to 70˚C. involved in the biosynthesis of phtiocerol dimycocerosates (PDIM). These lipids play a major role in the virulence of this microbial pathogen [START_REF] Astarie-Dequeker | Phthiocerol dimycocerosates of M. tuberculosis participate in macrophage invasion by inducing changes in the organization of plasma membrane lipids[END_REF] . Our approach combines a twobody dihydrofolate reductase (DHFR) scaffolding for selecting in frame DNA sequences from a random library of the fragmented ppsC gene, with the split-GFP technology [START_REF] Cabantous | In vivo and in vitro protein solubility assays using split GFP[END_REF] to identify soluble candidates centered onto the ACP domain (Figure 3a). The main advantage of our approach lies in the efficiency of the DHFR step to filter-out ~95% of the constructs present in the original DNA library and the use of an inverse PCR step to select for fragments encompassing a specific region or domain of a given protein. The implementation of the split-GFP assay to distinguish colonies on the basis of their fluorescence intensities completes this highly reliable and efficient strategy. We picked a total of 96 clones with fluorescence intensity levels varying from bright to faint depending on the solubility of the expressed fragment. Activity assays were carried out in the presence of the most soluble and compact ACP fragment 2042-2188 using either PptT or its closest homologue Sfp from Bacillus subtilis. We then visualized differences in the migration of the ACP domain on a 10% native polyacrylamide gel supplemented with 2.5 M urea (Figure 3b). Knowing that the reaction catalyzed by Sfp is magnesium-25 or manganese-dependent [START_REF] Mofid | Structurebased mutational analysis of the 4'-phosphopantetheinyl transferases Sfp from Bacillus subtilis: carrier protein recognition and reaction mechanism[END_REF] whereas the human AcpS X-ray structure revealed the presence of calcium in its active site [START_REF] Parris | Crystal structures of substrate binding to Bacillus subtilis holo-(acyl carrier protein) synthase reveal a novel trimeric arrangement of molecules resulting in three active sites[END_REF] , we then explored the role of all three divalent metal ions onto the activity of PptT. No activation of the ACP domain was detected in the presence of 10 mM CaCl 2 with either PptT or Sfp. When replacing CaCl 2 with MnCl 2 , activation of the ACP was noticeable after 30 min incubation, only in the presence of PptT. At 10 mM MgCl 2 , approximately half of the ACP domain was activated by the transfer of the Ppant moiety after 5 and 20 minutes incubation with PptT and Sfp, respectively. In conclusion, Mg 2+ resulted in the highest activity among the divalent ions tested followed by Mn 2+ , while Ca 2+ has no effect on the activity of the enzyme activity.

Discussion

Endogenous or exogenous ligands are often needed to stabilize a protein of interest in a particular conformation to carry out its function. Hosts such as E. coli have a great reservoir of co-factors that may be requisitioned for the correct folding and stability of the nascent protein. Although co-factors are produced in relatively small amounts compared to the overexpressed protein, early detection assay with sub-picomolar sensitivity, such as the one provided by the split-GFP system, can be very helpful to identify those proteins amenable to downstream functional and structural characterization. Optimizing the amount of soluble protein expressed can be attained using a systematic truncation strategy to cherry pick the best candidates.

In this study, we have applied the split-GFP system to the 4'-phosphopantetheinyl transferase (PptT) from M. tuberculosis, an enzyme required for growth and persistence of the bacteria in vivo [START_REF] Leblanc | 4'-Phosphopantetheinyl Transferase PptT, a New Drug Target Required for Mycobacterium tuberculosis Growth and Persistence In Vivo[END_REF] . PptT is responsible for the covalent transfer of the P-pant group of CoA to a conserved serine residue onto the acyl carrier protein (ACP) domain of various type I PKS. This post-translational modification converts PKS into functional holo enzymes that play a key role in the biosynthesis of various lipids in the cell envelope [START_REF] Chalut | The nonredundant roles of two 4'-phosphopantetheinyl transferases in vital processes of Mycobacteria[END_REF] . The pptT gene consists of 681 bp with a GC content of ~66% similar to all mycobacterial species. pptT also contains a total number of ten rare leucine, arginine and proline codons (http://nihserver.mbi.ucla.edu/RACC/). Despite the limited number of tRNA for rare codons in the BL21(DE3) E. coli strain, expression levels remained high.

Nevertheless, E. coli expression of PptT using pET vectors was hampered by solubility issues that caused the protein to partition exclusively into inclusion bodies, even after cleavage of the MBP solubility enhancer (Figure 1). To the opposite, its closest homologue, the surfactin synthetase-activating enzyme (Sfp) from B. subtilis, yields ~10 mg of pure protein from a 400 ml BL21(DE3) cell culture [START_REF] Mofid | Crystallization and preliminary crystallographic studies of Sfp: a phosphopantetheinyl transferase of modular peptide synthetases[END_REF] .

We took advantage of the weak AnTet-inducible promoter of the GFP11 vector [START_REF] Cabantous | Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein[END_REF] to investigate the solubility levels of an ensemble of forty-eight protein constructs in vivo, corresponding to the full-length enzyme along with N-and C-terminally truncated variants. Surprisingly, the full-length PptT appeared to be the most soluble protein construct after sequential induction in vivo (Figure 2). Although the in vitro experiments followed the same trend, aggregation of the protein over time ultimately led to its precipitation within hours. In an attempt to evaluate the contribution of the cellular cofactors Mg 2+ and CoA on the stability of PptT in vitro, a series of experiments using the DSF method was performed. High quality and reproducible fluorescent melting curves with Tm values of 50˚C could be measured only when CoA and MgCl 2 were maintained in a large molar excess over PptT, at concentrations equivalent to the purification conditions. Essentiality of these two cofactors for PptT to carry out its function was also demonstrated using a soluble fragment encompassing the ACP domain of the polyketide synthase PpsC (Figure 3). Transfer of the P-pant moiety onto the ACP domain could be observed in the present of Mg 2+ and, to a lesser extent, in the presence of Mn 2+ .

Surprisingly, activity of the B. subtilis Sfp was not detected in the presence of Mn 2+ , a result not in accordance with the work published by Mofid and colleagues [START_REF] Mofid | Structurebased mutational analysis of the 4'-phosphopantetheinyl transferases Sfp from Bacillus subtilis: carrier protein recognition and reaction mechanism[END_REF] , although the reaction mixture was incubated at 37˚C instead of 30˚C in our assay.

Fluorescence levels of clones obtained from N-terminally truncated constructs are similar to the full-length enzyme (Figure 2), thus indicating that residues at the Nterminal end do not play a critical role on the stability of the enzyme. We then generated a structure-based sequence alignment between structural homologues, the B. subtilis Sfp (PDB code 1QR0, [START_REF] Reuter | Crystal structure of the surfactin synthetase-activating enzyme sfp: a prototype of the 4'phosphopantetheinyl transferase superfamily[END_REF] ) and human AcpS (PDB code 2c43, [START_REF] Bunkoczi | Mechanism and substrate recognition of human holo ACP synthase[END_REF] ), that we further extended to

PptT with respect to the conservation of sequence motifs with key residues important for the activity and stability of CoA (Figure 4a). In the Sfp X-ray structure, N-terminal residues 2 to 7 form the outer most-strand of a three-stranded antiparallel β-sheet, at the opposite side of CoA (Figure 4b). This N-terminal β-strand also interacts with the 26residue long C-terminal region through hydrogen bond interactions that involve the mainchain nitrogen and oxygen atoms of Ile3 and Gly5. Tyr4 also contributes to the stabilization of the adjacent helix through interactions with Arg57. Disrupting all these interactions would have a dramatic impact onto the stability of the enzyme. In the human AcpS structure, the N-terminal domain is mostly disordered and unstructured with the first residue identified at position 8 in the X-ray structure and strand β1 starting at position 17 (Figure 4c). We then anticipated that the N-terminal region of PptT adopts an unstructured conformation similar to the one observed for the human AcpS, in accordance with the sequence alignment presented in Figure 4a. To the opposite, deletion of C-terminal residues has a dramatic effect on the solubility of PptT, as illustrated by the fluorescence drop to 75% for the truncated variant missing the C-terminus residue L227.

In the Sfp and AcpS structures, this highly variable and poorly structured region wraps around the α/β core of the protein (Figure 4b,c). Stabilization occurs through numerous hydrophobic and van der Waals contacts and a constellation of hydrogen bond interactions that involve the main-chain and side-chain atoms of residues all along the Cterminal region. At this stage, considering the low level of amino-acid sequence similarity between the three enzymes, questions about the role of L227 onto the stability of PptT are difficult to answer as no or X-ray structure is yet available. In the short run, we are confident that our efforts to overcome this major solubility bottleneck will help determining the three-dimensional structure of PptT. In the long run, the highthroughput screening of small molecule libraries will serve as a basis for the structurebased rational design of inhibitors with confirmed activity against M. tuberculosis.

5

 5 20 ml centrifugal down to 2 ml prior to injection into a HiLoad 16/60 Superdex 200 (GE Healthcare) pre-equilibrated with buffer B (50 mM Tris pH 8.0, 50 mM NaCl, 10 mM MgCl 2 , 50 µM CoA).ACP fragment cloning and purification.The ppsC gene from Mycobacterium tuberculosis was cloned into the NdeI/SpeI sites of a pET26b plasmid (Novagen, primers. DNA fragmentation, cloning of the DNA library into the DHFR ORF filter and the split-GFP solubility reporter[START_REF] Cabantous | In vivo and in vitro protein solubility assays using split GFP[END_REF] were conducted as previously described[START_REF] Pedelacq | Experimental mapping of soluble protein domains using a hierarchical approach[END_REF] . Briefly, a 400 to 850 bp DNA library was created by mechanical shearing of the PCR amplified ppsC gene. Blunt fragments were ligated in a StuI-digested insertion DHFR pET vector to select for inframe fragment in the presence of 6 μg/ml trimethoprim. Inverse PCR was performed with fragments ligated into the pTET ColE1 GFP 11 vector using phosphorylated forward (primers. After transformation into chemically competent BL21(DE3) pET GFP1-10 cells, ninety-six individually picked clones were grown overnight at 30°C in a 96-well tissue culture plate containing 7.5% glycerol in LB-Kan-Spec medium prior to sequencing. DNA sequences were analyzed using BioEdit® software and aligned onto the full-length parent gene to determine the exact boundaries. Based on the in vitro solubility assays[START_REF] Cabantous | In vivo and in vitro protein solubility assays using split GFP[END_REF] , fragments were color-coded black, dark gray, and light gray, where the black side of the spectrum identifies the least soluble ACP fragments and the light gray side corresponds to the top 25% most soluble ones. A DNA fragment encompassing the ACP domain and linker regions of PpsC was amplified by conventional PCR and was cloned into the NdeI/BamHI restriction sites of a pET28a plasmid (Novagen, Madison, WI) using GATATACATATGCATGACTCGGCGGCCCGCAAAA and AATTCGGATCCTGACTCGCCTCGCGTCGCAGCTT as forward and reverse primers, respectively. The resulting plasmid was transformed into chemically competent BL21(DE3) E. coli cells. Cells expressing the ACP fragment were grown to OD 600 ~ 0.6, induced with 0.25 mM IPTG for 4 h at 30ºC, pelleted by centrifugation at 4000 g for 30 min and stored at -80°C. The frozen pelleted cells were resuspended in 40 ml of buffer A and lysed by sonication (6 cycles of 30 s pulse, 50% amplitude, power 5) prior to centrifugation at 45,000 g for 30 min. 4 ml of Talon Superflow beads (Talon resin, Clontech, Palo Alto, CA) conditioned in buffer A were added to the supernatant for 5 min prior to centrifugation at 1,000 g for 5 min. The unbound fraction was removed and the beads were washed twice with 50 ml of buffer A then 30 ml of buffer A supplemented with 10 mM imidazole to eliminate non-specific binding to the beads. The N-terminally His-tagged protein was eluted with 5 ml of buffer A containing 250 mM imidazole. The protein elution sample was resolved on a 4-20% gradient SDS-PAGE gel and stained using a Coomassie® Blue R-250 staining solution. The protein solution was stored at -20°C after dialysis overnight against 25 mM Tris pH 8.0, 25 mM NaCl with 10 % glycerol. DSF experiments. Fluorescence of the SYPRO® Orange Protein Gel Stain (Invitrogen, USA) was measured using the CFX96™ real-time PCR detection System (Bio-Rad, Hercules, CA) from 20°C to 70°C in 0.3°C increments of 3 seconds. PptT was concentrated to 400 μM in the presence of buffer A (50 mM Tris pH 8.0, 50 mM NaCl, 10 mM MgCl 2 , 50 µM CoA) prior to a 10-fold dilution with either buffer A or buffer B (50 mM Tris pH 8.0, 50 mM NaCl). 20 μl mixtures containing 2 μl PptT 40 μM and 1 μl Sypro Orange 200X were placed in 96-well clear-bottom Hard-Shell ® 96-Well Skirted PCR Plates, Low-Profile (BioRad). Addition of 17 μl of buffer A or B completes the reaction mixture. Each condition was realized in triplicate. Melting temperatures were then calculated by the BioRad CFX Manager software. Activity assays. PptT at 50 nM was incubated at 30°C with 10 μM of ACP domain of PpsC. 20 μM CoA, 25 mM DTT and 10 mM MgCl2 were also present for the reaction to take place. Reaction was then stopped at different times with the addition of 100 mM EDTA. The same experiment was conducted in parallel using Sfp from B. subtilis (NEB, Ipswich, MA) at the same concentration instead of PptT. Samples were then loaded on a 10% polyacrylamide gel supplemented with 2.5 M urea. Positive and negative controls for the transfer of the Ppant moiety consisted in the incubation at 30°C with the same reactants for one hour with or without the presence of 200 nM Sfp as the only enzyme, respectively.

  PptT variants. Single amino-acid truncation at the Nterminal or C-terminal extremity of a protein of interest often has a dramatic effect onto the solubility level of the expressed variant11; 21 . With this in mind, we explored the expression and solubility levels of an ensemble of 48 constructs in vivo (Figure2a,b), corresponding to the full-length PptT along with N-and C-terminally truncated variants, using the split-GFP based assay developed by Cabantous and colleagues[START_REF] Cabantous | Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein[END_REF] . In this system, two separately inducible expression systems are present in the same E. coli cell, one tetracycline-inducible vector bearing the small GFP11 tag N-terminally fused to the protein of interest while the large complementary fragment GFP1-10 is located onto a separate IPTG-inducible expression vector. After sequential induction, only soluble GFP11 fusions can spontaneously bind to GFP1-10, thus restoring the fluorescence of the full-length GFP. In this system, fluorescent complementation is directly proportional to the amount of soluble protein present in the living cells or purified from cell crude extracts, with detection levels within the sub-picomole range. In vivo, the highest fluorescence levels could be attained for the full-length enzyme and N-terminally truncated variants whereas truncation of the last C-terminal residue L227 was sufficient to negatively impact the fluorescence intensity of the colonies (Figure2b). To confirm this result, an in vitro split-GFP complementation kinetic assay was performed by adding a large excess of GFP1-10 to the N-terminally and C-terminally truncated variants in the soluble fraction. After fifteen hours of complementation at room temperature, fluorescence intensity measured for the Δ227 deletion mutant was reduced by ~75% compared to the full-length enzyme (Figure2c). Fluorescence intensity levels measured for the other six C-terminally truncated variants, from position 226 to position 220, remained low and comparable. Both CoA and MgCl 2 are essential for the stability of PptT. In vivo expression and solubility levels of the recombinant His-PptT-GFP11 fusion were assessed by comparing the fluorescence intensities of co-induced and sequentially induced E. coli cell cultures,

  The low initial fluorescence background and the high signal-to-noise fluorescence ratios have facilitated an accurate measurement of the transition midpoint (Tm) values. High Tm values around 50˚C were measured in the presence of CoA and MgCl 2 at a final concentration of 50 μM and 10 mM, respectively, corresponding to the purification conditions. Diluting PptT so that CoA concentration was reduced to trace amount (0.5 μM final) and a 25-fold molar excess of MgCl 2 (100 μM final) over PptT profoundly impacted the stability of the full-length enzyme as no Tm values could be measured.In vitro enzymatic activity of PptT.PptT catalyzes the covalent transfer of the 4'phosphopantetheine (P-pant) group from CoA onto the ACP domain of various type I polyketide synthases (PKS)15; 17 . Issues related to heterologous host expression, folding and solubility of these large-size multi-domain enzymes forced us to identify a more compact and stand-alone soluble fragment encompassing the functional ACP domain. We used our recently published domain trapping strategy[START_REF] Pedelacq | Experimental mapping of soluble protein domains using a hierarchical approach[END_REF] to generate a 400-850 bp fragment library centered onto the ACP domain of PpsC, a 2188 amino-acid enzyme

Figure 1 .

 1 Figure 1. SDS-PAGE of soluble (S) and pellet (P) fractions of PptT constructs of E. coli BL21(DE3) cell cultures carrying a pET28-PptT plasmid allowing expression of PptT (1) with no 6His tag, (2) with N-terminal and (3) C-terminal 6His tag, (4) with Nterminally fused SUMO and (5) MBP proteins. Molecular weight markers (Mw) are also indicated.

Figure 2 .

 2 Figure 2. PCR-directed truncations of pptT and split-GFP complementation reaction. (a) Schematic representation of the 48 constructs aligned onto the PptT aminoacid sequence. Fragments are organized in groups of 6 with identical N-terminal positions and decreasing C-terminal positions. (b) Solubility screen of E. coli BL21(DE3) cells expressing the corresponding fragments in fusion with GFP11 following sequential complementation with GFP1-10. Most soluble fragments correspond to the full-length and N-terminally truncated constructs. (c) Superimposition of progress curves for complementation of PptT variants as a function of the fluorescence intensity versus time in minutes.

Figure 3 .

 3 Figure 3. Activation time-course of the ACP domain by PptT and the Sfp fromBacillus subtilis. (a) Schematic representation of the "domain trapping" strategy that integrates a DHFR filter to eliminate the out-of-frame constructs and a split-GFP solubility screen to identify those soluble ones. 450-850bp DNase I generated library of fragments was inserted at position 86/87 of the DHFR. Inverse PCR focused sub-libraries of fragments helped identifying a soluble fragment (indicated by an arrow) centered onto the ACP domain of the M. tuberculosis polyketide synthase PpsC. We used the color scheme gray -light gray -bright gray, to distinguish between the least and most soluble ACP fragments. (b) The transfer of the P-pant group from CoA to the ACP domain of PpsC is visualized on a 10% native polyacrylamide gel supplemented with 2.5 M urea in the presence of CaCl 2 (left), MnCl 2 (middle) and MgCl 2 (right) at different time intervals(5, 10, 15, 20, 30 and 60 min). Negative (-) and positive (+) controls correspond to the inactivated and activated forms of the ACP domain, respectively.

Figure 4 .

 4 Figure 4. Sequence alignment of PptT with known homologues. (a) Structure-based sequence alignment of the B. subtilis Sfp (Uniprot code P39135) and the human AcpS (Uniprot code Q9NRN7) extended to PptT with respect to the conservation of sequence motifs with key residues important for the activity and interactions with CoA. Secondary structure elements deduced from the X-ray structure of Sfp (PDB code 1QR0) and the human AcpS (PDB code 2C43) are shown at the bottom of the aligned sequences in light and dark gray, respectively. Sequence similarities are highlighted in red; sequence identities are shown as white letters on a red background. Strictly conserved catalytic residues D114 and E157 are indicated with brown triangles. The figure was produced with ESPript 30 . Ribbon representation of the X-ray structure of the B. subtilis Sfp (b) and the human AcpS (c). CoA is shown in a ball-and-stick representation. N-terminal and Cterminal ends are colored in green and orange in the X-ray structures and the sequence alignment. The side-chains of residues Tyr4 and Arg57 are indicated.

  Figure 1

  Figure 4

Acknowledgments

This work was supported by European Structural Funds (FEDER) and the Région Midi-Pyrénées (MYCA, grants 34249 and 09005193; TUBCAN, grant 11052698) and the Agence Nationale de la Recherche (XPKS-MYCO, grant 09-BLAN-0298-01)

Supplementary Table 1. PCR primers used for pptT truncations with


C Mouchache

V Griseri

N Saidi-Amroun

G Teyssèdre

S Mouaci

M Saidi

Dynamic of a Space Charge in Gamma-Irradiated Cross-Linked Polyethylene (XLPE)

The aim of the present work is to study the dynamics of the space charge in cross-linked polyethylene (XLPE) irradiated by gamma rays. For this purpose, film samples peeled from cables have been irradiated with gamma rays at total dose up to 100 kGy, in air at room temperature. To investigate the impact of the irradiation on the electrical behavior, space charge measurements were achieved in ambient conditions after the irradiation using Pulsed Electro Acoustic (PEA) method. The space charge measurements on the irradiated XLPE under an electric field of 40 kV/mm show the development of twoheterocharge regions adjacent to the electrodes. This is in mark contrast with the behavior of un-irradiated XLPE with space charge pattern dominated by charge injection. The charge accumulated during the polarization step persists during the relaxation and is stable after 1 hour.

I. INTRODUCTION

Cross-linked polyethylene (XLPE) is one of the most used materials in power cable industry because of his good dielectric and thermal properties. Space charges play an important role in the electrical behavior of XLPE, the reason why so many works are carried out to understand the origin and the impact of the formation of space charge on insulation reliability [START_REF] Teyssedre | Charge distribution and electroluminescence in cross-linked polyethylene under dc field[END_REF][START_REF] He | Space charge behavior in XLPE cable insulation under ac stress and its relation to thermo-electrical aging[END_REF]. However, the space charge in XLPE is still not fully understood and many processes can be involved in generation and accumulation of charges. Though the presence of space charge in XLPE under nuclear radiation environment has been investigated in some researches [START_REF] Fu | The effect of gamma irradiation on space charge behaviour and dielectric spectroscopy of low-density polyethylene[END_REF][START_REF] Kojima | Measurement of residual space charge distribution in γ-irradiated polyethylene[END_REF], the gross of the work is on the impact of field, temperature and material compounding, especially in the perspective of developing DC insulations. Ionizing radiation certainly constitutes a threat regarding space charge properties and insulation endurance. Indeed, when a polymeric insulation is submitted to an ionizing irradiation environment such as space environment for aircrafts or nuclear reactor for power systems, its dielectric and mechanical properties are altered due to chain scission, crosslinking and oxidation [START_REF] Fu | The effect of gamma irradiation on space charge behaviour and dielectric spectroscopy of low-density polyethylene[END_REF][START_REF] Sessler | Charge dynamics in irradiated polymers[END_REF].

In this paper, we investigate the impact of gamma irradiation on electrical behavior of XLPE. The experimental results consist on charge distribution in cross-linked polyethylene (XLPE) obtained by the pulsed electro-acoustic (PEA) method and the result show remarkable difference between irradiated and non-irradiated XLPE. Focus is given on the dynamic and origin of charges generated in the bulk of XLPE materials during polarization under dc field.

II. EXPERIMENTAL SET-UP

A. XLPE Samples

The cross-linked polyethylene (XLPE) was chosen as test material in the present study, since available directly from real power cables. Indeed, in the frame of the European project Artemis [START_REF] Fothergill | Electrical, microstructural, physical and chemical characterization of HV XLPE cable peelings for an electrical aging diagnostic data base[END_REF][START_REF] Teyssedre | Semi-quantitative analysis of photoluminescence in thermoelectrically aged cables: II-Analysis of a population of cables[END_REF], HVAC cables with 15 mm thick insulation were produced, and some sections of the produced cables were submitted to electrical and thermal aging in various conditions. Also, the cables were submitted to a wealth of electrical and structural characterization in previous works [START_REF] Fothergill | Electrical, microstructural, physical and chemical characterization of HV XLPE cable peelings for an electrical aging diagnostic data base[END_REF][START_REF] Teyssedre | Semi-quantitative analysis of photoluminescence in thermoelectrically aged cables: II-Analysis of a population of cables[END_REF] to reveal the impact of aging. Samples were used later after the project to investigate ageing under various electrical, thermal and radiation conditions [START_REF] Griseri | Dynamic of space charge in electron-beam irradiated cross-linked polyethylene[END_REF][START_REF] Tzimas | Effect of electrical and thermal stressing on charge traps in XLPE cable insulation[END_REF]. XLPE samples used in the present work were taken from rolls produced in the frame of the project, having thickness of 150 µm and width of 80 mm and stored in ambient conditions.

The sample were exposed to gamma irradiation in air at room temperature. The irradiation was made using 60 Co source with a dose rate of 23 Gy/min, and a total dose up to 100 kGy. We noticed a slight change in color after the irradiation: the samples then become more yellowish. A. Charlesby et al reported on similar yellowing of polyethylene irradiated with gamma rays [START_REF] Charlesby | Atomic Radiation and Polymers[END_REF]. Thermal ageing also produced yellowing on Artemis cables, presumably due to degradation of the antioxidant [START_REF] Teyssedre | Semi-quantitative analysis of photoluminescence in thermoelectrically aged cables: II-Analysis of a population of cables[END_REF].

B. Space charge measurements

Pulsed Electro-Acoustic -PEA measurements were achieved using a test cell provided by TechImp, Italy, suitable for the 150 µm thick samples used here. Experiments were achieved in air in an oven at a controlled temperature of 25°C. Pulsed voltages of 250 V amplitude, 8 ns width and 2 kHz repetition rate were used throughout the voltage cycle.

The sample was placed in sandwich configuration between the electrodes of the PEA cell, being ground Al electrode and semiconductor (carbon black doped polymer) as HV electrode. The space charge profiles were recorded in 20 s intervals, under the application of dc field of 40 kV/mm with positive polarity voltage for 1 hour followed by relaxation under 0 V for 1 hour. The voltage rising/falling rate was 1 kV/mm/s for electric field to reach the desired level.

III. RESULTS

A. Space charges in non-irradiated XLPE

Fig. 1 shows the charge developed in non-irradiated XLPE during polarization using DC field of 40 kV/mm for 1 hour. All profiles shown in this work are with 3 min interval time recording. As reported in previous work [START_REF] Mouchache | Space charge in electron-beam irradiated cross-linked polyethylene (XLPE): Charge implantation and release from the bulk[END_REF], the dominant mechanism observed on the recorded profiles for nonirradiated XLPE is the injection of charges followed by trapping near the electrode, forming homocharge. We clearly observe an injection of negative charge from the ground electrode, and the injection and charge accumulation processes are continuous during 1 hour stressing time. Indeed, the amount of negative charge grows slightly over time. It is also noticed that a small amount of positive charge was injected from the anode. However, the peak detected does not evolve with time. The influence charge on the ground electrode decreases over time, whereas the one at the HV electrode increases, due to the influence of the negative injected that creates positive image charges at both electrodes. The dominance of negative charges in the space charge profiles of XLPE was reported by Tzimas et al [START_REF] Tzimas | The effect of dc poling duration on space charge relaxation in virgin XLPE cable peelings[END_REF] for the same kind of samples taken from the Artemis program. It was also shown that steady state charging state could take several hours to be reached. It is therefore clear that the 1 h charging time used here is not enough to reach a steady state situation regarding charge build-up. Fig. 2 shows the time evolution of the space charge distribution when the applied field is removed. A negative charge is detected in the bulk of the sample and it seems to be spread homogeneously in all the insulation. The amount of negative charge decreases slowly during 1 hour. The positive induced charges on both electrodes represent the image of the negative charges trapped in the bulk.

Fig. 3 shows the distribution of the electric field all long the thickness. The electric field decreases slightly in the bulk with the increase of charging time, the electric field decreases from 50 kV/mm into 45 kV/mm in the end of polarization. The local electric field appear to be nearly homogeneously spread into the insulation.

B. Space charge in irradiated XLPE

The measurement results of space charge distribution in XLPE irradiated by gamma rays with a total dose of 100 kGy are represented in Fig. 4 for 1 hour stressing time under applied DC field of 40 kV/mm. The profile obtained before DC stressing reveals that there is no detectable amount of charge in the material, at least as net charge is concerned.

Upon DC stressing, negative and positive charge regions are gradually developed from the first moments of the application of external stress and the amount of heterocharge increases rapidly and continually during this period. The charge clouds on the profiles are adjacent to the anode for negative charges and to the cathode for positive ones. It is also noticeable that the charged regions are well symmetrical in the shape of the peak with the same maximum values of charge density.

The peak of influence charge recorded on the anode side shows a clear though slight increase in time. The variation is greater, or at least clearer at the cathode owing to the fact that the grounded electrode is adjacent to the piezoelectric sensor, hence the charge is better resolved than at the anode. The space charge profiles recorded during relaxation of the charge (0V applied) are presented in Fig. 5. The charges accumulated during the polarization step decrease slightly in 1 hour time. The maximum value in positive charge drops from 35 C/m 3 to 30 C/m 3 (14%). The decrease in the negative charge peak amplitude is faster than for the positive one, the drop in peak amplitude being from -35 C/m 3 to -25 C/m 3 (28%). These variations are reflected on the image charge peaks.

IV. DISCUSSION

The formation of large amount of negative and positive heterocharges in gamma-irradiated XLPE is a completely new behavior compared to the non-irradiated XLPE. Such a difference in the charge distribution is obviously related to the impact of the energy deposited by gamma ray in the material by causing reconfiguration of the electrical properties and presumably of the structure of XLPE. When the injection of charge from electrodes is the apparent mechanism responsible of space charge build-up in non-irradiated XLPE (Fig. 1,2), in the gamma irradiated XLPE the source of charges is apparently from the bulk. With the same stress applied on the non-irradiated and irradiated XLPE the charge developed in virgin XLPE is an order of magnitude less than in irradiated XLPE. In the irradiated sample the charge injection process was not observed or at least the heterocharge setting at the interface is much more important than the injected charge.

Compared to irradiation by an electron beam, where primary electrons were deposited and the deposited dose was not homogeneous along the thickness [START_REF] Mouchache | Space charge in electron-beam irradiated cross-linked polyethylene (XLPE): Charge implantation and release from the bulk[END_REF], gamma-rays are much more penetrating, producing homogeneous irradiation. As seen in Fig. 4, no charge was detected prior to voltage application.

The main question is on the origin of the charges being accumulated. Irradiation may produce electron-hole pairs or ion pairs into the material that can subsequently be driven toward the electrodes under application of the field. As there is no mass transfer across the interfaces, the ions could be accumulated near the interfaces. Fig. 6 provides an analysis of the charge decay kinetics with distinguishing the behavior of positive and negative charges. Charge, taken in absolute value, was integrated in the regions indicated in the caption. Though there is a somewhat higher amount of positive charges than of negative ones, the decay kinetics are similar. The interpretation in terms of ions movement would be consistent with the relative slow charge build up and with the fact that symmetrical charge patterns are observed. The net charge build-up could be explained by the movement of one polarity ions causing depletion on one side and accumulation on the other side. Radiation may also produce ionisable molecules or groups that could be dissociated under the effect of field and end with the same space charge features as with pre-existing ions.

A second possibility is that an interface layer with enhanced oxidation is present near both electrodes. The coloring of cables indeed reveals that chemical modification of the material has occurred. Fu et al [START_REF] Fu | The effect of gamma irradiation on space charge behaviour and dielectric spectroscopy of low-density polyethylene[END_REF] reported on very different space charge features depending whether gamma-irradiation was achieved in air or in neutral atmosphere. The space charge profile after irradiation at 100 kGy in air was much more complicated than when irradiating in vacuum and nitrogen at same dose. Resorting to low density polyethylene, oxidation was recognized as one of the main mechanisms inducing changes by the irradiation [START_REF] Kostoski | Effects of charge trapping in gamma irradiated and accelerated aged low-density polyethylene[END_REF][START_REF] Chen | Influence of radiation environments on space charge formation in γ-irradiated LDPE[END_REF]. Even analyzing the shape of space charge peak charge could be a way to identify the oxidized region [START_REF] Chen | Charge formation and decay in γirradiated low-density polyethylene[END_REF].

Oxidation could have two consequences: the formation of a polymer region near the surface with different electrical conductivity than in the bulk, and/or the formation of blocking layers for electronic carriers travelling through a sample with enhanced bulk conductivity due to irradiation. In the first case, symmetrical space charge patterns would be expected, and given the sign of the charge, the conductivity would be lower in the oxidized regions. The time for charge release would be slower than for build-up as the driving field is lower. In the second case, there is no a priori reason to get symmetrical charge patterns, both in charge amount and in build-up kinetics.

V. CONCLUSION Gamma-irradiation effects on space charge build-up was investigated in 150 µm thick cable peelings taken from HVAC cables. With such irradiation with 100 kGy dose achieved in air, substantial heterocharge build-up was observed at the two interfaces. The singularity in respect to previous works is the symmetrical features of the space charge being developed, considering the amount of charges, the peak shape and the build-up and decay kinetics. The behavior observed here could be related to the formation of an oxidized region near the interfaces. The electrical conductivity gradient introduced by such layer could be a reason for the obtained space charge patterns.

Fig. 1 .

 1 Fig. 1. Space charge profile recorded on a non-irradiated XLPE, during 1 h of polarisation under 40 kV/mm [12]. Profiles shown were obtained in 3 min interval time.

Fig. 2 .Fig. 3 .

 23 Fig.2. Space charge profile recorded on a non-irradiated XLPE, during 1 h of relaxation in Volt-off[START_REF] Mouchache | Space charge in electron-beam irradiated cross-linked polyethylene (XLPE): Charge implantation and release from the bulk[END_REF].

Fig. 4 .

 4 Fig. 4. Space charge profile recorded on a XLPE sample pre-irradiated with gamma ray. During 1 h of polarization under 40 kV/mm. The profile obtained prior to DC polarization is shown as bold red line.

Fig. 5 .

 5 Space charge profiles recorded on a XLPE pre-irradiated by gamma ray, during 1h of relaxation after polarization for 1h under 40 kV/mm.

Fig. 6 .

 6 Fig.6. Kinetic of integrated charge decay, with separation of positive and negative charges. The charge was integrated in the region from 10 µm to 105 µm for positive charges and 105 µm to 142 µm for negative ones.

ACKNOWLEDGMENT

The authors are grateful to the Director and staff (especially M. R. Yefsah) of the Nuclear Research Centre of Algiers for the provision of the irradiation facility. The work is supported by French-Algerian Tassili research program in the frame of Partenariat Hubert Curien (PHC).