International Journal of Biological Macromolecules 62 (2013) 549–556
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Cloning and heterologous expression of a thermostable pectate lyase from Penicillium occitanis in Escherichia coli Naourez Damak a,b , Salma Abdeljalil a,b , Aida Koubaa a,b , Sameh Trigui a,b , Malika Ayadi a,b , Hèla Trigui-Lahiani a,b , Emna Kallel a,b , Nadia Turki a,b , Lamia Djemal a,b , Hafeth Belghith a,b , Noomen Hadj Taieb a,b , Ali Gargouri a,b,∗ a Laboratoire de Valorisation de la Biomasse et Production des Protéines chez les Eucaryotes, Centre de Biotechnologie de Sfax, Route Sidi Mansour Km 6, PO Box 1177, 3018 Sfax, Tunisia b University of Sfax, Tunisia
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Article history: Received 27 August 2013 Received in revised form 11 October 2013 Accepted 11 October 2013 Available online 16 October 2013 Keywords: Pectate lyase Heterologous expression Medium composition
a b s t r a c t The entire pectate lyase cDNA (Pel1) of Penicillium occitanis was cloned from a cDNA bank and sequenced. The ORF exhibited a great homology to Penicillium marneffei and conservation of all features of fungal pectate lyases such as the barrel structure with “eight right-handed parallel -helix” architecture. The structure modeling also showed the interesting resemblance with thermostable pectate lyases since several specific residues were also shared by Pel1 and these thermostable enzymes. Having shown that the enzyme retains its activity after endoH-mediated deglycosylation, we investigated its expression in Escherichia coli BL21 using the pET28-a vector. This expression was shown to be optimum when cells were induced at room temperature in 2YT medium rather than at 37 ◦ C and LB medium. In such conditions, the recombinant protein was apparently produced more in soluble form than as inclusion bodies. The effect of NaCl concentration was investigated during the binding and elution steps of recombinant His-tagged enzyme on MagneHis Ni-particles. The purified enzyme was shown to retain its thermo-activity as well as a great tolerance to high concentration of NaCl and imidazole. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Plant pathogenic fungi use cellulases and pectinases to penetrate plant tissues during colonization [1]. Pectate lyases, otherwise known as pectate trans-eliminases, catalyze the cleavage of deesterified pectin, which is a major component of the primary cell walls of many higher plants [2]. Biotechnologically, pectinases are of great interest to food and juice industries [3,4] and textile industries [5]. There is a growing need to express individual pectinases in bacterial, yeast and fungal hosts. Expression level of pectate lyases in engineered fungi is low and the enzymes are difficult to purify [6–8]. Escherichia coli has been the “work-horse” of gene expression for many years as it is the best characterized host and the first-line system for producing recombinant proteins [9]. Several different host-vector systems are readily available in this organism which is simple to cultivate, growing rapidly and the recovery
∗ Corresponding author at: Laboratory of Biomass Valorisation and Production of Proteins in Eukaryotes, Centre of Biotechnology of Sfax, Route Sidi Mansour Km 6, PO Box 1177, 3018 Sfax, Tunisia. Tel.: +216 98938347; fax: +216 74 874 449. E-mail address: [email protected] (A. Gargouri). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.10.013
of recombinant proteins is relatively straightforward, particularly with the use of affinity tags. However, the systematic conversion of genetic information into a biologically active protein is constantly confronted to fundamental problems of protein folding. Several recombinant proteins are indeed not produced in their native state; instead, they aggregate into a biologically inactive form. Although this aggregation reaction has some practical advantages, in vitro refolding of recombinant proteins, after solubilization of cellular aggregates, is sometimes an empiric and random process. Several studies pointed out the effect of the medium and culture composition and conditions on the yield of soluble proteins and inclusion bodies produced [10]. In a previous work [11], the pel1 pectate lyase of Penicillium occitanis was produced, purified and shown to be thermoactive at 60 ◦ C. In this work, we isolated the Pel1 cDNA and complete gene. The protein structure was predicted by modeling and compared to other thermostable enzymes. The cDNA was then expressed in E. coli using the pET28 vector. This heterologous production was explored by varying different parameters in order to increase the production level of the recombinant protein in a soluble form, enabling to escape all the refolding procedures. The IMAC purification conditions were also optimized by varying the NaCl concentration during the binding and elution steps.
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2. Materials and methods 2.1. Strains The P. occitanis strain CT1 derived from the wild-type strain CL100 of P. occitanis after a single round of nitrous acid mutagenesis from the wild type strain CL100 of P. occitanis [12]. The strains CL100 and CT1 were deposited at the culture collection of the Biotechnology Center of Sfax-Tunisia under references CTM10246 and CTM10496, respectively. These strains were propagated on potato dextrose agar (PDA) and stored as a spore suspension in 20% glycerol at −80 ◦ C until further use. E. coli strains: Top 10 F ((F lacIq Tn10 (TetR)) mcrA (mrr-hsdRMS-mcrBC) 80lacZ M15 lacX74 recA1 araD139 (ara-leu)7697 galU galK rpsL (StrR) endA1 nupG; Invitrogen) was used as a host for plasmids propagation. BL21 (DE3): F– ompT gal dcm lon hsdSB (rB- mB-) (DE3 [lacI lacUV5-T7 gene 1 ind1 sam7nin5]) was used for the production of recombinant proteins. ER1647 (recD−, mcrA−, hsdRMS/mcrB/mrr, tet+) was used as a host for cDNA bank construction and phage propagation. BM25.8 (supE44, thi D(lac–proAB) [F traD36, proAB+ lacIqZPM15] limm434 (KanR) P1 (CamR) hsdR (rk12− mk12−) was used to convert phage into plasmid DNA.
[14]. (2) Differential hybridization was carried out using two labeled probes consisting of radioactive cDNA constructed by reverse transcription of RNA: the first non-induced one is from the glucose grown CL100 strain, the induced one derived from pectingrown CT1 strain. The probes were labeled using the Random Primed DNA Labeling kit (rediprimeTM II; Amersham). Hybridization was performed according to the manufacturer’s protocols (–N+; Amersham) at 65 ◦ C. The blots were twice washed in 2× SSC, 0.1% SDS at ambient temperature and then twice at 65 ◦ C. After screening, DNA of interesting phages were recovered and transformed into BM25.8 E. coli cells, where the cre recombinase liberated plasmids from the phage, through its activity on lox boxes, as recommended by the manufacturer. 2.6. Sequencing and sequence analysis The nucleotide sequence was carried out using the BigDye® Terminator v3.1 Cycle Sequencing Kit in the ABI PRISM 3100Avant Genetic Analyzer (Applied Biosystems Inc.) with universal and reverse primers. The pel1 cDNA sequence reported in this article has been submitted to the GenBank under accession number HM171486. DNA sequence analyses were performed using the BioEdit and Blast programs.
2.2. Vectors
2.7. Cloning in pET28 and expression of pel
The pMOSblue (Amersham) and the pGEM-T vector (Promega) were used for the cloning of PCR fragments; MOS Elox plasmid (Amersham) was used for the construction of the cDNA library; pMOS Elox derived from the excision of the MOS Elox after infection of the BM 25.8 strain. The pET28-a(+) was used to express the pectate lyase cDNA in E. coli under the control of the T7 promoter. The pectate lyase was fused at its N-terminal end to His Tag.
Potato dextrose agar (PDA, Merck Co.) was used for the propagation and the storage of fungal strains. These strains were also grown in a modified Mandels liquid medium described in [13] for 5 days at 30 ◦ C and 150 rpm. The modified medium composition was the following (g/l): 2.0 K2 HPO4 , 1.4 (NH4 )2 SO4 , 0.3 MgSO4 , 1.0 yeast extract, 5 sodium phtalate, 10 of either citrus pectin or glucose as carbon sources; finally 0.3 g/l CaCl2 was added after autoclaving. Luria broth (LB: 0.5% Yeast extract, 1% Bacto-trypton, 0.5% NaCl) and 2YT (1.7% Bacto-tryptone, 1% Yeast extract, 0.5% NaCl) media were used for the cultivation of bacterial strains. Antibiotics were added to these media (30 g ml−1 kanamycin; 100 g ml−1 ampicillin; 12.5 g ml−1 tetracycline) as required.
The pel1 cDNA was amplified with PfuUltraTM high-fidelity DNA polymerase (fermentas) using pel1 specific primers: sense one (5 -GGGATCCATGGCATCCGTCAGCT) and anti-sense (5 ATCGATACTAGAAA GACAACGTCTGTCC), corresponding to the mature form of the pectate lyase enzyme and verified by sequencing. The DNA fragment was released from pGEM-T and inserted in the expression vector pET28a(+), giving rise to the fusion between the 6×His-Tag sequence of the vector and the Pel1 sequence. The resultant recombinant clone was verified by PCR and enzymatic digestion. The gene is thus placed under the control of the bacteriophage T7 promoter that can be specifically transcribed by the T7RNA polymerase, whose gene is present in the bacterial chromosome of E. coli BL21 strain, under the control of lacUV5 promoter which is inducible by IPTG. The pET28-pel1 plasmid was transferred into E. coli BL21 that was cultivated at 37 ◦ C and 200 rpm to reach an absorbance (at 600 nm) of 0.6. The conditions of induction were optimized by varying different parameters: the culture medium (LBK and 2YTK), the temperature (room temperature and 37 ◦ C), the concentration of inducer (0.4, 0.8, 1.6 mM of IPTG) and time after induction (0.5, 2, 3, 4 and 24 h). Note that after induction, the cultures were continued at room temperature and 150 rpm to reduce the inclusion bodies formation.
2.4. Construction of the cDNA bank
2.8. Protein extraction and purification
Total RNA was extracted from the pectin induced mycelia of 5-day-old cultures of CT1. From the total RNA, polyA+ mRNA were isolated using an oligo-dT cellulose column and following the manufacturer’s protocols (Amersham), converted into cDNA, then into double stranded DNA, ligated with MOSelox phage arms and finally packaged into phage particles. These reconstituted phages served for transformation of ER1647 E. coli cells and the construction of phage bank.
Intracellular fraction was prepared from biomass recovered from different cultures conditions, as follows: after centrifugation of the cultured cells at 12,000 rpm, the cells pellet was frozen at −80 ◦ C. The frozen cells were grinded with alumina in PBS buffer containing 1 mM PMSF. The lysate was centrifuged at 3000 rpm for 10 min, the pellet (containing non-lysed cells and cell debris) was discarded while the supernatant (named crude extract) is centrifuged at 13,000 rpm for 20 min. The pellet contained the inclusion bodies was named “P” while the supernatant contained the soluble recombinant enzyme was named “S” fraction. The latter one was used for the purification of the Pel enzyme. 1 ml of S fraction was supplemented by NaCl, reaching various concentrations (0, 100, 250 and 500 mM NaCl) and mixed with 30 l of MagneHis Ni-particles at room temperature for 2 min with
2.3. Media and growth conditions
2.5. Screening of the cDNA library and plasmid manipulations All plates of the cDNA bank were transferred on N+ filters in duplicate. Two hybridizations were carried out: (1) specific hybridization to V2-4, an amplified fragment of pectin lyase gene
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gentle shaking. After incubation, the tube was placed in a magnetic stand for 1 min to allow the MagneHis Ni-particles to be captured by the magnet, and the supernatant was removed. The particles were washed three times with the binding/wash buffer then purified protein was eluted using various imidazole concentration (dissolved in the respective NaCl concentrations) and subjected to SDS/PAGE. 2.9. Gel electrophoresis and protein quantification The proteins were loaded on a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) after boiling the sample for 5 min as described by Laemmli [15]. Proteins bands were visualized by Coomassie brilliant blue R-250 (Biorad) staining. Low-molecular-weight proteins marker (Amersham) was used as standard. The proteins concentration was determined using Bradford’s method with bovine serum albumin as the standard [16]. 2.10. Enzyme activity assay Pectate lyase activity was determined according to the method described by Pitt [17] in which 5 ml PGA solution (1%, w/v in 20 mM glycine buffer at pH 9), 1 ml CaCl2 (0.01 M) and 1 ml of diluted enzyme were used. The total volume was adjusted to 10 ml with water and incubated for 2 h at 60 ◦ C. The reaction was then stopped by the addition of 0.6 ml ZnSO4 ·7H2 O (9%, w/v) followed by 0.6 ml NaOH (0.5 M). The precipitate was centrifuged at 3000 × g for 10 min. A volume of 3 ml TBA (Thiobarbituric acid) at 0.04 M was added to 5 ml of the supernatant, followed by 1.5 ml HCl (0.1 M) and 0.5 ml water. The mixture was heated in boiling water bath for 30 min and then cooled. Absorbance was measured at 550 nm. In the control tubes, on the other hand, the enzyme was added after the addition of ZnSO4 and NaOH. One unit of Pel was defined as the amount of enzyme causing a change in absorbance of 0.01 under the described assay conditions. The optimum temperature for Pel1 activity was determined by incubating the enzyme preparation with PGA (polygalacturonic acid) at temperatures 50, 60 and 70 ◦ C (at pH 9.0). Pel1 activity was determined using the standard assay described above. 2.11. Multiple sequence alignment and protein modeling analysis The ClustalW2 program from the website (http://www.ebi. ac.uk/Tools/msa/clustalw2/) was used to align the P. occitanis pectate lyase 1 (pel1) (ADK25712.1) with the pectate lyase A (XP 002149699.1) from Talaromyces marneffei which had the highest sequence identity and four available pectate lyase sequences, presenting the following accession number: XP 749213.1 from Aspergillus fumigatus, XP 001265635.1 from Neosartorya fischeri, EPS31113.1 from Penicillium oxalicum and NP 638163.1 from Xanthomonas campestris strain. The tertiary structure of the deduced amino acid sequence of P. occitanis pectate lyase 1 was predicted by homology modeling using the Swiss-Model Server [1] using the crystal structure of pectate lyase II from X. campestris (PDB: 2qy1A) as template [18]. The parameters and prediction quality of the modeled structure was evaluated using the program SPDBV v. 4.01. 3. Results and discussion 3.1. Isolation and analysis of P. occitanis pectate lyase cDNA We have already selected a particular mutant from the fungus P. occitanis which is characterized by a constitutive hyper-secretion of pectinases and only pectinases [12]. This constitutive overexpression of pectinases is transcriptionally regulated in the CT1 mutant since the mRNA of several pectinases (polygalacturonase,
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pectin lyase and pectate lyase) were over expressed not only on Pectin, the inducing substrate, but also on glucose, usually known as repressor of pectinolytic gene expression [14]. To better understand the behavior of this mutant, we need further knowledge on pectinolytic enzymes and their encoding genes. In a previous work, we purified a thermostable pectate lyase and showed that it is highly expressed either on glucose or pectin, confirming the constitutive hyper expression in the CT1 strain. In this work, we planned to clone the cDNA of this pectate lyase (named Pel1) of P. occitanis from a cDNA bank. The cDNA library was constructed using 2 g of mRNA according to the manufacturer’s instructions of the cDNA Synthesis System kit (Amersham). Library screening was done with the RT-PCR amplified fragment V2-4, as a probe of 430 bp long, belonging to the pectinase family since it exhibited a strong similarity with fungal pectin lyases [14]. In parallel, differential hybridization of the library was carried out using two radio labeled probes: the first one is represented by the cDNA of CL100 strain which has been cultivated on glucose while the second one derived from pectin or glucose-grown CT1 strain (see Section 2). We have to recall that pectinases of the CL100 wild type strain are completely repressed by glucose while they are highly produced by CT1 either on glucose or pectin since CT1 is a constitutive mutant which hyperproduces pectinases even on glucose. Using both hybridization strategies, several positive phages were isolated and converted into plasmid clones thanks to the cre-lox system. The nucleotide sequence of different inserts was then blasted to conventional banks and three of them showed strong homologies with pectate lyase (clones 1, 12 and 31). Clone 31 derived from the first and second hybridization strategy while clones 1 and 12 derived from the differential screening. The clone 1 contained the longest open reading frame, which started few bases before the ATG initiator codon and ended after the STOP codon (Fig. 1). The mature form of the pectate lyase is identical to the N-terminal amino acid sequence, already determined on the purified enzyme [11]. This mature form starts after “Lys Arg” residues (Fig. 1), the known Kex2-like peptidase signaling for secreted proteins which are expressed as pre-pro-enzymes [19]. This suggests that a Kex2like enzyme should exist in P. occitanis, as it is the case in other filamentous fungi where the dibasic Kex2-peptidase sites predominate as target sequences in secretory proteins [20]. This implies that Pel1 should be expressed as a pre-pro-enzyme, being secreted after the removal of the pre–pro sequence. The gene was also isolated by screening the genomic bank, already constructed in the laboratory [21]. It contains 4 short introns, presenting roughly the same signature than other introns already determined in other genes (data not shown).
3.2. Analysis of multiple alignment and prediction of P. occitanis pel 1 three-dimensional structure ClustalW alignment of pectate lyase 1 (Pel1) from P. occitanis with known pectate lyase amino acid sequences highlighted several consensus regions including those which are known to confer thermostability as well as the three highly conserved catalytic residues: Lys (K)184, Arg (R) 217 and Arg (R) 222 (Fig. 2). Sanchez-Torres et al. have performed site-directed-mutagenesis studies on family 1 pectin lyase A (PL1A) from Aspergillus niger to identify amino acid residues critical for catalysis and stability: substituting Arg 236 with alanine or lysine rendered the enzyme completely inactive and mutagenesis of Arg 176 and Lys 239 severely affected catalysis [18]. It is worth to note that the comparison between the structures of pectin and pectate lyases has indicated that both lyases most likely descended from a common ancestor enzyme as both of
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tatcaagaaaacaGaaccaaaacccgcaaaaagatcgagaaaacatcttcatcATGAAG M K GCC A GCA A GCT A GGT G GCC A ATC I ATA I CTG L TCC S TCC S AAC N GCC A TGG
W
TTC F CCG P ACA T GGT G GTG V ACC T ATT I GTC V AAG K AAC N CAC H GCA A AAA
K
ATC I GCT A GGC G ACC T ACC T CAG Q GGC G AAG K GTG V AAT N GAC D AAC N GCC
ACA T GAA E TAC Y GCG A GGC G ACA T AAG K GAA E CTT L GTC V AAG K TTC F TCC
GCT A CAA Q GCA A ACT T AAT N GCT A GAC D GCT A GCC A TGG W GAC D ATA I CTA
CTC L CTT L AGC S ACC T GCC A GAT D TCG S TCA S GCC A ATC I TAC Y ACC T ATC
GCC A GTC V CTC L GTC V AAA K CAG Q AGT S AAC N AAT N GAC D TAC Y GTC V GGC
CTC L AAG K AAC N TCC S AAA K ATC I GCC A GTG V GGA G CAC H GAC D TCC S CAC
GCT A AGA R GGC G AGC S GTC V AGA R AAA K GTT V GAT D GTC V GGC G AAC N TCG
AGC S GCA A GGC G TAC Y ATC I CCT P TTG L ATC I GCT A GAC D CTC L AGC
S
AGC S TCC S ACA T GCT A TTC F GGT G GTC V CGT R ATT I GTC V CTC L TAC
Y
TAT Y GTC V ACC T GCT A GTG V AAC N AAT N AAC N GGT G TCC S GAC D ATC
I
GCT A AGC S GGT G CTC L TCC S AAC N TTT F TTG L GTC V TCT S CTC L CAC
H
TTG L GAC D GGC G GCA A GGC G ACC T GGA G GGT G CAA Q GAC D ACC T GAC
D
GCT A GCA A GCC A ACC T ACT T AGC S ATT I ATT I TAC Y CGC R CAC H CAC
H
GAC AAC AAC GGC GCA GAG A S L I G H S D N N G A E GGC CAC CTA CGC GTA ACC CAA AAC AAC AAT CAC TGG TAC G H L R V T Q N N N H W Y AAC TCT CGC ACC CCT TCC ATC CGT TTC GGC ACG GGC CAC N S R T P S I R F G T G H AAC AGC TAC TTT GAC CAA GTC AAC GAC GGC ATC AAT ACC N S Y F D Q V N D G I N T GGC GCA CAG GTG CTT GTG CAG TCG AAT GTG TTT GTG GGG G A Q V L V Q S N V F V G AAG CCG CTT TAT TCG ACT GAT GAT GGA TAT GCA GTT GCA K P L Y S T D D G Y A V A AAT GAT TTC GGG AGT GGA AGT AAT GAG GCG CTG GCG GGG N D F G S G S N E A L A G ACG TCT GTG CCG TAT AGT TAT TCG TTG TTG GGG AGT GGG T S V P Y S Y S L L G S G AAA GCT GCT GTT GTG AGC ACT GCT GGA CAG ACG TTG TCT K A A V V S T A G Q T L S TTTATGTAGCTGTATAGTATGGAAGCCATTTGAGGCTATGATGATCGGAGG
GAC ACC D T AAC ATT N I GTA TAC V Y CGC GAC R D TCA AGC S S ACG GAT T D ACG CTG T L AAT GTT N V TTC TAG F END TTTTTGAGTGGTGGACTGGGGAGGATATGTTTACCTATA
Fig. 1. Sequence of cDNA and deduced amino acid sequence of Penicillium occitanis pectate lyase 1 (Pel1). N terminal sequence of pel1 is underlined; Cleavage site of signal peptide (K R residues) are shaded; Predicted site of glycosylation (N X T/S) are boxed.
them are constituted by a barrel of parallel beta strands coiled in a right handed helix [22]. The identification of conserved residues, common to all thermostable pectate lyases, in the Pel1 sequence correlates with our previous studies showing the biochemical features of this novel thermoactive fungal pectate lyase [10]. In fact, the optimum temperature and pH value required for maximal enzymatic activity were found to be 60 ◦ C and pH 9.0, respectively. Moreover, pectate lyases require Ca2+ for their activity [23]. Ca2+ is essential for the activity of all pectate lyases, including Pel1 of P. occitanis which requires 1 mM CaCl2 for its optimal activity [11] Xiao et al. compiled several pectate lyases sequences and showed the conservation of three aspartic acids which coordinated the
calcium [24]. By comparison, we identified these three conserved calcium binding sites in Pel1: Asp (D) 131, 160 and 164 (Fig. 2). Building a structural model for P. occitanis pectate lyase 1 needs the use of the best template presenting the highest identity between the two sequences. Although Pel1 amino acid sequence showed high degree of homology with fungal pectate lyase, unfortunately, none of them has been crystallized, nor have their 3D structure been established. The only known structures are of bacterial Pectate lyases. Recently, three structures of pectate lyase II from X. campestris have been deposited in PDB: the wild type PLXc (PDB ID 2QX3), the single mutated pectate lyase R236F (PDB ID 2QXZ) and the double mutated pectate lyase A31G/R236F (PDB ID 2QY1) [18]. The
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Fig. 2. Multiple alignment of protein sequences of P. occitanis Pel 1 (ADK25712.1), Talaromyces marneffei (XP 002149699.1), Aspergillus fumigatus (XP 749213.1), Neosartorya fischeri (XP 001265635.1), Penicillium oxalicum (EPS31113.1) and the thermostable pectate lyase Xanthomonas campestris (NP 638163.1). The numbering of each sequence starts from the initiation codon. Conserved catalytic sites are shown with asterisks, conserved calcium binding sites are indicated with dark arrows, the core structure of the parallel -helix (vWiDH region) is boxed with dark-interrupted line and sites conserved in pectate lyases are boxed with dark-full line.
highest identity score of 30% was obtained between P. occitanis Pel 1 (Tm , 60 ◦ C) and the template 2QY1 chainA (the above mentioned double mutated PL of X. campestris), which was therefore used to generate a structural model by Swiss-Model Automatic Modeling Mode (http://swissmodel.expasy.org/) (Fig. 3). The generated model showed the typical “eight right-handed parallel -helix” architecture, which has been described for the majority of pectate lyase belonging to family 1. The core structure of the parallel -helix is formed within the “VWiDH” region (see alignment). Note that the helical structure was previously found to enhance the catalytic activity of related pectate lyases [11]. Xiao et al. compiled several thermolabile and thermoresistant pectate lyases and determined consensus residues of thermoresistant ones [24]. Among these residues, they demonstrated the importance of the G31 and F236 in the thermoresistance through directed mutagenesis. They studied four variants of X. campestris: the wild-type, the single mutants A31G, R236F and the double mutant A31G/R236F. They established the Tm (melting temperature: the temperature at which 50% of the protein is unfolded) of
the four protein variants to be 48, 47.5, 54, and 53 ◦ C, respectively [24]. They showed that single amino acid substitutions affect either the thermostability or the catalytic activity of the enzyme [24].
3.3. The endoH- deglycosylated pectate lyase retains its activity The inspection of the amino-acid sequence revealed two sites of N-glycosilation (Fig. 1). In fact, both sites are juxtaposed in the same sequence NNTS, as given by the N-Glyc server, knowing that the N-glycosylation consensus is “Asn-X-Ser/Thr”: the first one is therefore NNT and the second is NTS (Fig. 1). Our previous results indicated that the native Pel1 (pectate lyase purified from CT1 mutant) was indeed glycosylated [11]. Here, we show that the endoglycosidase H treatment did not affect its activity. Indeed, the activity of the purified native pectate lyase passed from 520 units/ml to 513 units/ml after 1 h of treatment by endoH N-glycosidase. This result encouraged us to express the protein in bacterial system, where no glycosylation is thought to occur.
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Fig. 3. Modeling prediction of P. occitanis Pel1 and 3D-visualation with SwissPdbViewer 4.0 (modeled residues range from 7 to 290 based on 2qy1A template ˚ from Xanthomonas campestris pectate lyase A31G/R236F variant: 1.90 A).
3.4. Optimization of the conditions of heterologous expression pectate lyase gene in E. coli The cDNA was sub-cloned in the vector pET28a(+), as in Section 2, while the control strain was transformed by the empty vector. After 2 h post-induction by 1 mM IPTG, the biomass was recovered from both cultures, from which we extracted intracellular proteins (referred as crude extract) and divided into soluble “S” and pellet “P” fractions. Fig. 4A shows clearly that the recombinant protein, with a MW of 36 kDa, was produced at high amount in the P fraction of the recombinant strain, suggesting that it is highly expressed as inclusion bodies. Note that no difference can be observed between the two soluble fractions on the same SDS–PAGE (Fig. 4A). Nevertheless, the dosage of pectate lyase activity in the different fractions revealed interesting features. No activity was found in the fractions of the control strain while high amounts were found in either the crude extract CE or the S fraction of the recombinant strain. More precisely, starting with 150 ml culture, 332 units of Pel activity were recorded in CE, leading after centrifugation to the recovery of 504 units in the S fraction and only 33 units in the P fraction. This means firmly that the inclusion bodies (IB), contained in P fraction, were inactive despite their huge amounts (see Fig. 4A). Moreover and by the way, we note a better recovery of activity after centrifugation (compare 504–332 units), which suggests that probably some inhibitors have been discarded during the centrifugation. In order to optimize the conditions for producing the pectate lyase of P. occitanis in E. coli, we varied several parameters including the medium, the temperature, the induction time and the inducer concentration. The recombinant strain was therefore grown either on LB or 2YT medium. After induction with different concentrations of IPTG and culture during various times at room temperature, we recovered the crude extract as well as the P and the S fractions from each condition. The heterologous expression of proteins was assessed by SDS–PAGE and by dosage of the pectate lyase activity. On LB medium, after 4 h of induction, 0.4 mM IPTG was insufficient to reveal the recombinant protein while increasing the concentration of IPTG to 1.6 mM, a protein with a molecular weight of 36 kDa was observed. On 2YT medium, the hetrologous expression was improved at two levels: the concentration of 0.4 mM IPTG was sufficient and the induction time could be reduced to 2 h. This
Fig. 4. (A) SDS–PAGE gel electrophoresis (12%) of 20 g of intracellular proteins; with C: cell extract of the control strain carrying the empty vector; R: cell extract of the recombinant strain. After centrifugation, the intracellular proteins were divided in two fractions: S: supernatant representing the soluble proteins and P: pellet representing the inclusion bodies. The cultures of recombinant strains were performed on 2YT medium and the induction was carried out at 1 mM of IPTG for 3 h. (B) Profile comparison between intracellular proteins of 2YT and LB biomass, at two culture temperatures (RT and 37 ◦ C), migrated on SDS–PAGE gel electrophoresis (12%) of 20 g of proteins; with S: supernatant representing the soluble proteins and P: pellet representing the inclusion bodies.
behavior is different from the Pecate lyase of Aspergillus nidulans which was expressed in E. coli after 60 h of induction in presence of 0.5 mM IPTG [25]. Other works have reported on the great influence of yeast extract on the expression of recombinant proteins in E. coli [10]. Finally, in order to test the effect of temperature on the production level and to verify once again the difference between both media, we compared the expression on LB and 2YT at room temperature RT (about 25 ◦ C) and 37 ◦ C, while maintaining constants all the other parameters: 0.4 mM of IPTG, 150 rpm of erlenmeyers agitation and 3 h post induction. Fig. 4B shows the analysis of all S and P fractions from LB and 2YT culture conditions (RT and 37 ◦ C), Table 1 Comparison of the effects of culture conditions (the medium composition and the temperature) on the production of recombinant pectate lyase activity. LB
Tot Prot (mg/ml) Activity (units/ml)
2YT
37 ◦ C
RT
37 ◦ C
RT
3.96 4.5
4.24 14.42
5.36 7.43
7.99 41.26
Tot prot: intracellular proteins.
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Table 2 Effect of salt concentration in the binding buffer on protein recovery. 500 mM NaCl
PAL before dialysis (units) PAL after dialysis (units)
250 mM NaCl
300 mM imidazole
500 mM imidazole
1M imidazole
300 mM imidazole
500 mM imidazole
1M imidazole
5.4 7.3
0.5 3
0.1 0.2
4.1 9.2
0.2 0.3
0.2 0.3
where we can see less amounts of inclusion bodies in 2YT than in LB medium, on both culture temperature. In parallel, higher amounts of activity were recorded in the S fractions of 2YT than those of LB conditions with a clear advantage in favor of RT than 37 ◦ C culture condition, see Table 1. Therefore, all together, these results suggest that the recombinant enzyme was much more produced in soluble form than IB in 2YT than in LB and at RT than at 37 ◦ C. 3.5. Purification of the recombinant pectate lyase The optimized conditions were applied to purify the recombinant enzyme. 1 l of 2YT medium was inoculated by the recombinant strain, cultured on 37 ◦ C until reaching 0.6 as OD600 nm , then induced by 0.4 mM IPTG and incubated at room temperature with shaking at 150 rpm for 2 h. The intracellular extract was centrifuged at 13,000 × g and the supernatant was applied on MagneHis Niparticles. The binding of the His-tagged pectate lyase was firstly performed using two salt buffer (500 mM and 250 mM NaCl), then washed with 50 mM imidazole. The elution was therefore performed stepwise using increasing concentration of imidazole (50, 200 300 and 500 mM) in the two corresponding NaCl concentrations (Table 2). Interestingly, using 250 mM NaCl allowed us to elute the maximum of recombinant enzyme (9.2 units recovered
after dilaysis) at 300 mM imidazole while such concentration of imidazole was insufficient to elute all the activity when using 500 mM NaCl (only 7.3 units recovered after dialysis). Indeed, a second 500 mM imidazole step was needed to elute the remaining 3 units, compared to 0.3 units in the 250 mM condition. Moreover, even if the dialysis improved the recovery of the enzyme, it is worth to note that the recombinant enzyme was active in presence of 0.5 M NaCl and 300 mM imidazole, see Table 2, first column. Besides dosages, the electrophoresis analysis of the eluted fractions (from both 500 and 250 mM NaCl conditions) revealed a contamination of the recombinant protein with several E. coli proteins. This problem was resolved by lowering the NaCl concentration to either 100 or 0 mM. Indeed, after binding to the column, the elution was carried using various imidazole concentrations. In both conditions, the recombinant protein was eluted using only 200 mM imidazole. Fig. 5 shows the result obtained using 100 mM NaCl in the binding and elution buffer. A single and pure protein was eluted with 200 mM imidazole, at the right size of 36 kDa (Fig. 5). The recovered activity was of about 46 units, from a total of 504 units in the S fraction (see above), which means a recovery yield of slightly less than 10%. Finally, the purified and dialyzed fraction was tested for thermoactivity. The recombinant protein retained the same behavior with regard to temperature, being optimal at 60 ◦ C. Indeed, it exhibited the following activities: 25, 38 and 4 units/ml for the temperatures 50, 60 and 70 ◦ C. 4. Conclusion The cDNA of pectate lyase of P. occitanis was cloned from a cDNA bank. This cDNA was sub-cloned in the vector pET28a(+). In order to optimize the conditions for producing the pectate lyase of P. occitanis in E. coli several parameters were varied including the medium composition, the temperature, the induction time and the inducer concentration. Interestingly, we showed that the medium composition and the culture temperature greatly influence the mode of expression of our protein: it was much more expressed in soluble form on 2YT than on LB and at room temperature (about 25 ◦ C) than at 37 ◦ C. The IMAC purification method was optimized by varying the NaCl concentration during the binding and elution steps. 100 mM NaCl was the most convenient as it leads to the purification of a pure protein. This recombinant enzyme retained its thermo-active character since its optimum of activity was reached at 60 ◦ C. References
Fig. 5. Purification of the recombinant pectate lyase from E. coli. (A) Using 100 mM NaCl in binding and elution buffer. 1: eluted fractions (with 200 mM imidazole) were separated on a 12% SDS–polyacrylamide gel and stained by Coomassie blue. The arrow indicates the recombinant Pel1. M: protein markers.
[1] A. Collmer, N.T. Keen, Annual Review of Phytopathology 24 (1986) 383–409. [2] N.C. Carpita, D.M. Gibeaut, Plant Journal 3 (1993) 1–30. [3] D.R. Kashyap, P.K. Vohra, S. Chopra, R. Tewari, Bioresource Technology 77 (2001) 215–227. [4] W. Pilnik, F.M. Rombouts, Carbohydrate Research 142 (1) (1985) 93–105. [5] H.S.S. Sharma, Applied Microbiology and Biotechnology 26 (1987) 358–362. [6] J.A. Benen, H.C. Kester, L. Parenicová, J. Visser, Biochemistry 39 (50) (2000) 15563–15569. [7] L. González-Candelas, A. Cortell, D. Ramon, FEMS Microbiology Letters 126 (1995) 263–269.
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[8] W. Guo, L. González-Candelas, P.E. Kolattukudy, Archives of Biochemistry and Biophysics 323 (2) (1995) 352–360. [9] S.Y. Lee, Trends in Biotechnology 14 (1996) 98–105. [10] X.Y. Fu, Z.D. Wei, W.Y. Tong, Journal of Chemical Technology and Biotechnology 81 (2006) 1866–1871. [11] N. Damak, N. Hadj-Taieb, E. Bonnin, A. Ben Bacha, A. Gargouri, Process Biochemistry 46 (2011) 888–893. [12] N. Hadj-Taieb, M. Ayadi, S. Trigui, F. Bouabdallah, A. Gargouri, Enzyme and Microbial Technology 30 (2002) 662–666. [13] M. Mandels, J. Weber, Advances in Chemistry Series 95 (1969) 391–412. [14] M. Ayadi, S. Trigui, H. Trigui-Lahiani, N. Hadj-Taïeb, M. Jaoua, A. Gargouri, Biotechnology Letters 33 (2011) 1139–1144. [15] U.K. Laemmli, Nature 277 (1970) 680–685. [16] M.M. Bradford, Analytical Biochemistry 72 (1976) 248–254. [17] D. Pitt, in: W.A. Wood, S.T. Kellogg (Eds.), Methods in Enzymology, vol. 161, Academic Press, San Diego, 1988, pp. 350–354.
[18] P. Sanchez-Torres, J. Visser, J.A.E. Benen, Biochemical Journal 370 (2003) 331–337. [19] R.S. Fuller, A.J. Brake, J. Thorner, Science 246 (4929) (1989) 482–486. [20] T.P.G. Calmels, F. Martin, H. Durand, G. Tiraby, Journal of Biotechnology 17 (1991) 51–66. [21] H. Trigui-Lahiani, A. Gargouri, Gene 388 (2007) 54–60. [22] O. Mayans, M. Scott, I. Connerton, T. Gravesen, J. Benen, J. Visser, R. Pickersgill, J. Jenkins, Structure 5 (5) (1997) 677–689. [23] S.R. Herron, R.D. Scavetta, M. Garrett, M. Legner, F. Jurnak, Journal of Biological Chemistry 278 (2003) 12271–12277. [24] Z. Xiao, H. Bergeron, S. Grosse, M. Beauchemin, M.L. Garron, D. Shaya, T. Sulea, M. Cygler, P.C.K. Lau, Applied and Environmental Microbiology 74 (2008) 1183–1189. [25] Q.X. Zhao, S. Yuan, Y.L. Zhang, H. Zhu, F.M. Hang, World Journal of Microbiology and Biotechnology 23 (2007) 1057–1064.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Cloning and heterologous expression of a thermostable pectate lyase from Penicillium occitanis in Escherichia coli Naourez Damak a,b , Salma Abdeljalil a,b , Aida Koubaa a,b , Sameh Trigui a,b , Malika Ayadi a,b , Hèla Trigui-Lahiani a,b , Emna Kallel a,b , Nadia Turki a,b , Lamia Djemal a,b , Hafeth Belghith a,b , Noomen Hadj Taieb a,b , Ali Gargouri a,b,∗ a Laboratoire de Valorisation de la Biomasse et Production des Protéines chez les Eucaryotes, Centre de Biotechnologie de Sfax, Route Sidi Mansour Km 6, PO Box 1177, 3018 Sfax, Tunisia b University of Sfax, Tunisia
a r t i c l e
i n f o
Article history: Received 27 August 2013 Received in revised form 11 October 2013 Accepted 11 October 2013 Available online 16 October 2013 Keywords: Pectate lyase Heterologous expression Medium composition
a b s t r a c t The entire pectate lyase cDNA (Pel1) of Penicillium occitanis was cloned from a cDNA bank and sequenced. The ORF exhibited a great homology to Penicillium marneffei and conservation of all features of fungal pectate lyases such as the barrel structure with “eight right-handed parallel -helix” architecture. The structure modeling also showed the interesting resemblance with thermostable pectate lyases since several specific residues were also shared by Pel1 and these thermostable enzymes. Having shown that the enzyme retains its activity after endoH-mediated deglycosylation, we investigated its expression in Escherichia coli BL21 using the pET28-a vector. This expression was shown to be optimum when cells were induced at room temperature in 2YT medium rather than at 37 ◦ C and LB medium. In such conditions, the recombinant protein was apparently produced more in soluble form than as inclusion bodies. The effect of NaCl concentration was investigated during the binding and elution steps of recombinant His-tagged enzyme on MagneHis Ni-particles. The purified enzyme was shown to retain its thermo-activity as well as a great tolerance to high concentration of NaCl and imidazole. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Plant pathogenic fungi use cellulases and pectinases to penetrate plant tissues during colonization [1]. Pectate lyases, otherwise known as pectate trans-eliminases, catalyze the cleavage of deesterified pectin, which is a major component of the primary cell walls of many higher plants [2]. Biotechnologically, pectinases are of great interest to food and juice industries [3,4] and textile industries [5]. There is a growing need to express individual pectinases in bacterial, yeast and fungal hosts. Expression level of pectate lyases in engineered fungi is low and the enzymes are difficult to purify [6–8]. Escherichia coli has been the “work-horse” of gene expression for many years as it is the best characterized host and the first-line system for producing recombinant proteins [9]. Several different host-vector systems are readily available in this organism which is simple to cultivate, growing rapidly and the recovery
∗ Corresponding author at: Laboratory of Biomass Valorisation and Production of Proteins in Eukaryotes, Centre of Biotechnology of Sfax, Route Sidi Mansour Km 6, PO Box 1177, 3018 Sfax, Tunisia. Tel.: +216 98938347; fax: +216 74 874 449. E-mail address: [email protected] (A. Gargouri). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.10.013
of recombinant proteins is relatively straightforward, particularly with the use of affinity tags. However, the systematic conversion of genetic information into a biologically active protein is constantly confronted to fundamental problems of protein folding. Several recombinant proteins are indeed not produced in their native state; instead, they aggregate into a biologically inactive form. Although this aggregation reaction has some practical advantages, in vitro refolding of recombinant proteins, after solubilization of cellular aggregates, is sometimes an empiric and random process. Several studies pointed out the effect of the medium and culture composition and conditions on the yield of soluble proteins and inclusion bodies produced [10]. In a previous work [11], the pel1 pectate lyase of Penicillium occitanis was produced, purified and shown to be thermoactive at 60 ◦ C. In this work, we isolated the Pel1 cDNA and complete gene. The protein structure was predicted by modeling and compared to other thermostable enzymes. The cDNA was then expressed in E. coli using the pET28 vector. This heterologous production was explored by varying different parameters in order to increase the production level of the recombinant protein in a soluble form, enabling to escape all the refolding procedures. The IMAC purification conditions were also optimized by varying the NaCl concentration during the binding and elution steps.
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2. Materials and methods 2.1. Strains The P. occitanis strain CT1 derived from the wild-type strain CL100 of P. occitanis after a single round of nitrous acid mutagenesis from the wild type strain CL100 of P. occitanis [12]. The strains CL100 and CT1 were deposited at the culture collection of the Biotechnology Center of Sfax-Tunisia under references CTM10246 and CTM10496, respectively. These strains were propagated on potato dextrose agar (PDA) and stored as a spore suspension in 20% glycerol at −80 ◦ C until further use. E. coli strains: Top 10 F ((F lacIq Tn10 (TetR)) mcrA (mrr-hsdRMS-mcrBC) 80lacZ M15 lacX74 recA1 araD139 (ara-leu)7697 galU galK rpsL (StrR) endA1 nupG; Invitrogen) was used as a host for plasmids propagation. BL21 (DE3): F– ompT gal dcm lon hsdSB (rB- mB-) (DE3 [lacI lacUV5-T7 gene 1 ind1 sam7nin5]) was used for the production of recombinant proteins. ER1647 (recD−, mcrA−, hsdRMS/mcrB/mrr, tet+) was used as a host for cDNA bank construction and phage propagation. BM25.8 (supE44, thi D(lac–proAB) [F traD36, proAB+ lacIqZPM15] limm434 (KanR) P1 (CamR) hsdR (rk12− mk12−) was used to convert phage into plasmid DNA.
[14]. (2) Differential hybridization was carried out using two labeled probes consisting of radioactive cDNA constructed by reverse transcription of RNA: the first non-induced one is from the glucose grown CL100 strain, the induced one derived from pectingrown CT1 strain. The probes were labeled using the Random Primed DNA Labeling kit (rediprimeTM II; Amersham). Hybridization was performed according to the manufacturer’s protocols (–N+; Amersham) at 65 ◦ C. The blots were twice washed in 2× SSC, 0.1% SDS at ambient temperature and then twice at 65 ◦ C. After screening, DNA of interesting phages were recovered and transformed into BM25.8 E. coli cells, where the cre recombinase liberated plasmids from the phage, through its activity on lox boxes, as recommended by the manufacturer. 2.6. Sequencing and sequence analysis The nucleotide sequence was carried out using the BigDye® Terminator v3.1 Cycle Sequencing Kit in the ABI PRISM 3100Avant Genetic Analyzer (Applied Biosystems Inc.) with universal and reverse primers. The pel1 cDNA sequence reported in this article has been submitted to the GenBank under accession number HM171486. DNA sequence analyses were performed using the BioEdit and Blast programs.
2.2. Vectors
2.7. Cloning in pET28 and expression of pel
The pMOSblue (Amersham) and the pGEM-T vector (Promega) were used for the cloning of PCR fragments; MOS Elox plasmid (Amersham) was used for the construction of the cDNA library; pMOS Elox derived from the excision of the MOS Elox after infection of the BM 25.8 strain. The pET28-a(+) was used to express the pectate lyase cDNA in E. coli under the control of the T7 promoter. The pectate lyase was fused at its N-terminal end to His Tag.
Potato dextrose agar (PDA, Merck Co.) was used for the propagation and the storage of fungal strains. These strains were also grown in a modified Mandels liquid medium described in [13] for 5 days at 30 ◦ C and 150 rpm. The modified medium composition was the following (g/l): 2.0 K2 HPO4 , 1.4 (NH4 )2 SO4 , 0.3 MgSO4 , 1.0 yeast extract, 5 sodium phtalate, 10 of either citrus pectin or glucose as carbon sources; finally 0.3 g/l CaCl2 was added after autoclaving. Luria broth (LB: 0.5% Yeast extract, 1% Bacto-trypton, 0.5% NaCl) and 2YT (1.7% Bacto-tryptone, 1% Yeast extract, 0.5% NaCl) media were used for the cultivation of bacterial strains. Antibiotics were added to these media (30 g ml−1 kanamycin; 100 g ml−1 ampicillin; 12.5 g ml−1 tetracycline) as required.
The pel1 cDNA was amplified with PfuUltraTM high-fidelity DNA polymerase (fermentas) using pel1 specific primers: sense one (5 -GGGATCCATGGCATCCGTCAGCT) and anti-sense (5 ATCGATACTAGAAA GACAACGTCTGTCC), corresponding to the mature form of the pectate lyase enzyme and verified by sequencing. The DNA fragment was released from pGEM-T and inserted in the expression vector pET28a(+), giving rise to the fusion between the 6×His-Tag sequence of the vector and the Pel1 sequence. The resultant recombinant clone was verified by PCR and enzymatic digestion. The gene is thus placed under the control of the bacteriophage T7 promoter that can be specifically transcribed by the T7RNA polymerase, whose gene is present in the bacterial chromosome of E. coli BL21 strain, under the control of lacUV5 promoter which is inducible by IPTG. The pET28-pel1 plasmid was transferred into E. coli BL21 that was cultivated at 37 ◦ C and 200 rpm to reach an absorbance (at 600 nm) of 0.6. The conditions of induction were optimized by varying different parameters: the culture medium (LBK and 2YTK), the temperature (room temperature and 37 ◦ C), the concentration of inducer (0.4, 0.8, 1.6 mM of IPTG) and time after induction (0.5, 2, 3, 4 and 24 h). Note that after induction, the cultures were continued at room temperature and 150 rpm to reduce the inclusion bodies formation.
2.4. Construction of the cDNA bank
2.8. Protein extraction and purification
Total RNA was extracted from the pectin induced mycelia of 5-day-old cultures of CT1. From the total RNA, polyA+ mRNA were isolated using an oligo-dT cellulose column and following the manufacturer’s protocols (Amersham), converted into cDNA, then into double stranded DNA, ligated with MOSelox phage arms and finally packaged into phage particles. These reconstituted phages served for transformation of ER1647 E. coli cells and the construction of phage bank.
Intracellular fraction was prepared from biomass recovered from different cultures conditions, as follows: after centrifugation of the cultured cells at 12,000 rpm, the cells pellet was frozen at −80 ◦ C. The frozen cells were grinded with alumina in PBS buffer containing 1 mM PMSF. The lysate was centrifuged at 3000 rpm for 10 min, the pellet (containing non-lysed cells and cell debris) was discarded while the supernatant (named crude extract) is centrifuged at 13,000 rpm for 20 min. The pellet contained the inclusion bodies was named “P” while the supernatant contained the soluble recombinant enzyme was named “S” fraction. The latter one was used for the purification of the Pel enzyme. 1 ml of S fraction was supplemented by NaCl, reaching various concentrations (0, 100, 250 and 500 mM NaCl) and mixed with 30 l of MagneHis Ni-particles at room temperature for 2 min with
2.3. Media and growth conditions
2.5. Screening of the cDNA library and plasmid manipulations All plates of the cDNA bank were transferred on N+ filters in duplicate. Two hybridizations were carried out: (1) specific hybridization to V2-4, an amplified fragment of pectin lyase gene
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gentle shaking. After incubation, the tube was placed in a magnetic stand for 1 min to allow the MagneHis Ni-particles to be captured by the magnet, and the supernatant was removed. The particles were washed three times with the binding/wash buffer then purified protein was eluted using various imidazole concentration (dissolved in the respective NaCl concentrations) and subjected to SDS/PAGE. 2.9. Gel electrophoresis and protein quantification The proteins were loaded on a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) after boiling the sample for 5 min as described by Laemmli [15]. Proteins bands were visualized by Coomassie brilliant blue R-250 (Biorad) staining. Low-molecular-weight proteins marker (Amersham) was used as standard. The proteins concentration was determined using Bradford’s method with bovine serum albumin as the standard [16]. 2.10. Enzyme activity assay Pectate lyase activity was determined according to the method described by Pitt [17] in which 5 ml PGA solution (1%, w/v in 20 mM glycine buffer at pH 9), 1 ml CaCl2 (0.01 M) and 1 ml of diluted enzyme were used. The total volume was adjusted to 10 ml with water and incubated for 2 h at 60 ◦ C. The reaction was then stopped by the addition of 0.6 ml ZnSO4 ·7H2 O (9%, w/v) followed by 0.6 ml NaOH (0.5 M). The precipitate was centrifuged at 3000 × g for 10 min. A volume of 3 ml TBA (Thiobarbituric acid) at 0.04 M was added to 5 ml of the supernatant, followed by 1.5 ml HCl (0.1 M) and 0.5 ml water. The mixture was heated in boiling water bath for 30 min and then cooled. Absorbance was measured at 550 nm. In the control tubes, on the other hand, the enzyme was added after the addition of ZnSO4 and NaOH. One unit of Pel was defined as the amount of enzyme causing a change in absorbance of 0.01 under the described assay conditions. The optimum temperature for Pel1 activity was determined by incubating the enzyme preparation with PGA (polygalacturonic acid) at temperatures 50, 60 and 70 ◦ C (at pH 9.0). Pel1 activity was determined using the standard assay described above. 2.11. Multiple sequence alignment and protein modeling analysis The ClustalW2 program from the website (http://www.ebi. ac.uk/Tools/msa/clustalw2/) was used to align the P. occitanis pectate lyase 1 (pel1) (ADK25712.1) with the pectate lyase A (XP 002149699.1) from Talaromyces marneffei which had the highest sequence identity and four available pectate lyase sequences, presenting the following accession number: XP 749213.1 from Aspergillus fumigatus, XP 001265635.1 from Neosartorya fischeri, EPS31113.1 from Penicillium oxalicum and NP 638163.1 from Xanthomonas campestris strain. The tertiary structure of the deduced amino acid sequence of P. occitanis pectate lyase 1 was predicted by homology modeling using the Swiss-Model Server [1] using the crystal structure of pectate lyase II from X. campestris (PDB: 2qy1A) as template [18]. The parameters and prediction quality of the modeled structure was evaluated using the program SPDBV v. 4.01. 3. Results and discussion 3.1. Isolation and analysis of P. occitanis pectate lyase cDNA We have already selected a particular mutant from the fungus P. occitanis which is characterized by a constitutive hyper-secretion of pectinases and only pectinases [12]. This constitutive overexpression of pectinases is transcriptionally regulated in the CT1 mutant since the mRNA of several pectinases (polygalacturonase,
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pectin lyase and pectate lyase) were over expressed not only on Pectin, the inducing substrate, but also on glucose, usually known as repressor of pectinolytic gene expression [14]. To better understand the behavior of this mutant, we need further knowledge on pectinolytic enzymes and their encoding genes. In a previous work, we purified a thermostable pectate lyase and showed that it is highly expressed either on glucose or pectin, confirming the constitutive hyper expression in the CT1 strain. In this work, we planned to clone the cDNA of this pectate lyase (named Pel1) of P. occitanis from a cDNA bank. The cDNA library was constructed using 2 g of mRNA according to the manufacturer’s instructions of the cDNA Synthesis System kit (Amersham). Library screening was done with the RT-PCR amplified fragment V2-4, as a probe of 430 bp long, belonging to the pectinase family since it exhibited a strong similarity with fungal pectin lyases [14]. In parallel, differential hybridization of the library was carried out using two radio labeled probes: the first one is represented by the cDNA of CL100 strain which has been cultivated on glucose while the second one derived from pectin or glucose-grown CT1 strain (see Section 2). We have to recall that pectinases of the CL100 wild type strain are completely repressed by glucose while they are highly produced by CT1 either on glucose or pectin since CT1 is a constitutive mutant which hyperproduces pectinases even on glucose. Using both hybridization strategies, several positive phages were isolated and converted into plasmid clones thanks to the cre-lox system. The nucleotide sequence of different inserts was then blasted to conventional banks and three of them showed strong homologies with pectate lyase (clones 1, 12 and 31). Clone 31 derived from the first and second hybridization strategy while clones 1 and 12 derived from the differential screening. The clone 1 contained the longest open reading frame, which started few bases before the ATG initiator codon and ended after the STOP codon (Fig. 1). The mature form of the pectate lyase is identical to the N-terminal amino acid sequence, already determined on the purified enzyme [11]. This mature form starts after “Lys Arg” residues (Fig. 1), the known Kex2-like peptidase signaling for secreted proteins which are expressed as pre-pro-enzymes [19]. This suggests that a Kex2like enzyme should exist in P. occitanis, as it is the case in other filamentous fungi where the dibasic Kex2-peptidase sites predominate as target sequences in secretory proteins [20]. This implies that Pel1 should be expressed as a pre-pro-enzyme, being secreted after the removal of the pre–pro sequence. The gene was also isolated by screening the genomic bank, already constructed in the laboratory [21]. It contains 4 short introns, presenting roughly the same signature than other introns already determined in other genes (data not shown).
3.2. Analysis of multiple alignment and prediction of P. occitanis pel 1 three-dimensional structure ClustalW alignment of pectate lyase 1 (Pel1) from P. occitanis with known pectate lyase amino acid sequences highlighted several consensus regions including those which are known to confer thermostability as well as the three highly conserved catalytic residues: Lys (K)184, Arg (R) 217 and Arg (R) 222 (Fig. 2). Sanchez-Torres et al. have performed site-directed-mutagenesis studies on family 1 pectin lyase A (PL1A) from Aspergillus niger to identify amino acid residues critical for catalysis and stability: substituting Arg 236 with alanine or lysine rendered the enzyme completely inactive and mutagenesis of Arg 176 and Lys 239 severely affected catalysis [18]. It is worth to note that the comparison between the structures of pectin and pectate lyases has indicated that both lyases most likely descended from a common ancestor enzyme as both of
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tatcaagaaaacaGaaccaaaacccgcaaaaagatcgagaaaacatcttcatcATGAAG M K GCC A GCA A GCT A GGT G GCC A ATC I ATA I CTG L TCC S TCC S AAC N GCC A TGG
W
TTC F CCG P ACA T GGT G GTG V ACC T ATT I GTC V AAG K AAC N CAC H GCA A AAA
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ATC I GCT A GGC G ACC T ACC T CAG Q GGC G AAG K GTG V AAT N GAC D AAC N GCC
ACA T GAA E TAC Y GCG A GGC G ACA T AAG K GAA E CTT L GTC V AAG K TTC F TCC
GCT A CAA Q GCA A ACT T AAT N GCT A GAC D GCT A GCC A TGG W GAC D ATA I CTA
CTC L CTT L AGC S ACC T GCC A GAT D TCG S TCA S GCC A ATC I TAC Y ACC T ATC
GCC A GTC V CTC L GTC V AAA K CAG Q AGT S AAC N AAT N GAC D TAC Y GTC V GGC
CTC L AAG K AAC N TCC S AAA K ATC I GCC A GTG V GGA G CAC H GAC D TCC S CAC
GCT A AGA R GGC G AGC S GTC V AGA R AAA K GTT V GAT D GTC V GGC G AAC N TCG
AGC S GCA A GGC G TAC Y ATC I CCT P TTG L ATC I GCT A GAC D CTC L AGC
S
AGC S TCC S ACA T GCT A TTC F GGT G GTC V CGT R ATT I GTC V CTC L TAC
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TAT Y GTC V ACC T GCT A GTG V AAC N AAT N AAC N GGT G TCC S GAC D ATC
I
GCT A AGC S GGT G CTC L TCC S AAC N TTT F TTG L GTC V TCT S CTC L CAC
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TTG L GAC D GGC G GCA A GGC G ACC T GGA G GGT G CAA Q GAC D ACC T GAC
D
GCT A GCA A GCC A ACC T ACT T AGC S ATT I ATT I TAC Y CGC R CAC H CAC
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GAC AAC AAC GGC GCA GAG A S L I G H S D N N G A E GGC CAC CTA CGC GTA ACC CAA AAC AAC AAT CAC TGG TAC G H L R V T Q N N N H W Y AAC TCT CGC ACC CCT TCC ATC CGT TTC GGC ACG GGC CAC N S R T P S I R F G T G H AAC AGC TAC TTT GAC CAA GTC AAC GAC GGC ATC AAT ACC N S Y F D Q V N D G I N T GGC GCA CAG GTG CTT GTG CAG TCG AAT GTG TTT GTG GGG G A Q V L V Q S N V F V G AAG CCG CTT TAT TCG ACT GAT GAT GGA TAT GCA GTT GCA K P L Y S T D D G Y A V A AAT GAT TTC GGG AGT GGA AGT AAT GAG GCG CTG GCG GGG N D F G S G S N E A L A G ACG TCT GTG CCG TAT AGT TAT TCG TTG TTG GGG AGT GGG T S V P Y S Y S L L G S G AAA GCT GCT GTT GTG AGC ACT GCT GGA CAG ACG TTG TCT K A A V V S T A G Q T L S TTTATGTAGCTGTATAGTATGGAAGCCATTTGAGGCTATGATGATCGGAGG
GAC ACC D T AAC ATT N I GTA TAC V Y CGC GAC R D TCA AGC S S ACG GAT T D ACG CTG T L AAT GTT N V TTC TAG F END TTTTTGAGTGGTGGACTGGGGAGGATATGTTTACCTATA
Fig. 1. Sequence of cDNA and deduced amino acid sequence of Penicillium occitanis pectate lyase 1 (Pel1). N terminal sequence of pel1 is underlined; Cleavage site of signal peptide (K R residues) are shaded; Predicted site of glycosylation (N X T/S) are boxed.
them are constituted by a barrel of parallel beta strands coiled in a right handed helix [22]. The identification of conserved residues, common to all thermostable pectate lyases, in the Pel1 sequence correlates with our previous studies showing the biochemical features of this novel thermoactive fungal pectate lyase [10]. In fact, the optimum temperature and pH value required for maximal enzymatic activity were found to be 60 ◦ C and pH 9.0, respectively. Moreover, pectate lyases require Ca2+ for their activity [23]. Ca2+ is essential for the activity of all pectate lyases, including Pel1 of P. occitanis which requires 1 mM CaCl2 for its optimal activity [11] Xiao et al. compiled several pectate lyases sequences and showed the conservation of three aspartic acids which coordinated the
calcium [24]. By comparison, we identified these three conserved calcium binding sites in Pel1: Asp (D) 131, 160 and 164 (Fig. 2). Building a structural model for P. occitanis pectate lyase 1 needs the use of the best template presenting the highest identity between the two sequences. Although Pel1 amino acid sequence showed high degree of homology with fungal pectate lyase, unfortunately, none of them has been crystallized, nor have their 3D structure been established. The only known structures are of bacterial Pectate lyases. Recently, three structures of pectate lyase II from X. campestris have been deposited in PDB: the wild type PLXc (PDB ID 2QX3), the single mutated pectate lyase R236F (PDB ID 2QXZ) and the double mutated pectate lyase A31G/R236F (PDB ID 2QY1) [18]. The
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Fig. 2. Multiple alignment of protein sequences of P. occitanis Pel 1 (ADK25712.1), Talaromyces marneffei (XP 002149699.1), Aspergillus fumigatus (XP 749213.1), Neosartorya fischeri (XP 001265635.1), Penicillium oxalicum (EPS31113.1) and the thermostable pectate lyase Xanthomonas campestris (NP 638163.1). The numbering of each sequence starts from the initiation codon. Conserved catalytic sites are shown with asterisks, conserved calcium binding sites are indicated with dark arrows, the core structure of the parallel -helix (vWiDH region) is boxed with dark-interrupted line and sites conserved in pectate lyases are boxed with dark-full line.
highest identity score of 30% was obtained between P. occitanis Pel 1 (Tm , 60 ◦ C) and the template 2QY1 chainA (the above mentioned double mutated PL of X. campestris), which was therefore used to generate a structural model by Swiss-Model Automatic Modeling Mode (http://swissmodel.expasy.org/) (Fig. 3). The generated model showed the typical “eight right-handed parallel -helix” architecture, which has been described for the majority of pectate lyase belonging to family 1. The core structure of the parallel -helix is formed within the “VWiDH” region (see alignment). Note that the helical structure was previously found to enhance the catalytic activity of related pectate lyases [11]. Xiao et al. compiled several thermolabile and thermoresistant pectate lyases and determined consensus residues of thermoresistant ones [24]. Among these residues, they demonstrated the importance of the G31 and F236 in the thermoresistance through directed mutagenesis. They studied four variants of X. campestris: the wild-type, the single mutants A31G, R236F and the double mutant A31G/R236F. They established the Tm (melting temperature: the temperature at which 50% of the protein is unfolded) of
the four protein variants to be 48, 47.5, 54, and 53 ◦ C, respectively [24]. They showed that single amino acid substitutions affect either the thermostability or the catalytic activity of the enzyme [24].
3.3. The endoH- deglycosylated pectate lyase retains its activity The inspection of the amino-acid sequence revealed two sites of N-glycosilation (Fig. 1). In fact, both sites are juxtaposed in the same sequence NNTS, as given by the N-Glyc server, knowing that the N-glycosylation consensus is “Asn-X-Ser/Thr”: the first one is therefore NNT and the second is NTS (Fig. 1). Our previous results indicated that the native Pel1 (pectate lyase purified from CT1 mutant) was indeed glycosylated [11]. Here, we show that the endoglycosidase H treatment did not affect its activity. Indeed, the activity of the purified native pectate lyase passed from 520 units/ml to 513 units/ml after 1 h of treatment by endoH N-glycosidase. This result encouraged us to express the protein in bacterial system, where no glycosylation is thought to occur.
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Fig. 3. Modeling prediction of P. occitanis Pel1 and 3D-visualation with SwissPdbViewer 4.0 (modeled residues range from 7 to 290 based on 2qy1A template ˚ from Xanthomonas campestris pectate lyase A31G/R236F variant: 1.90 A).
3.4. Optimization of the conditions of heterologous expression pectate lyase gene in E. coli The cDNA was sub-cloned in the vector pET28a(+), as in Section 2, while the control strain was transformed by the empty vector. After 2 h post-induction by 1 mM IPTG, the biomass was recovered from both cultures, from which we extracted intracellular proteins (referred as crude extract) and divided into soluble “S” and pellet “P” fractions. Fig. 4A shows clearly that the recombinant protein, with a MW of 36 kDa, was produced at high amount in the P fraction of the recombinant strain, suggesting that it is highly expressed as inclusion bodies. Note that no difference can be observed between the two soluble fractions on the same SDS–PAGE (Fig. 4A). Nevertheless, the dosage of pectate lyase activity in the different fractions revealed interesting features. No activity was found in the fractions of the control strain while high amounts were found in either the crude extract CE or the S fraction of the recombinant strain. More precisely, starting with 150 ml culture, 332 units of Pel activity were recorded in CE, leading after centrifugation to the recovery of 504 units in the S fraction and only 33 units in the P fraction. This means firmly that the inclusion bodies (IB), contained in P fraction, were inactive despite their huge amounts (see Fig. 4A). Moreover and by the way, we note a better recovery of activity after centrifugation (compare 504–332 units), which suggests that probably some inhibitors have been discarded during the centrifugation. In order to optimize the conditions for producing the pectate lyase of P. occitanis in E. coli, we varied several parameters including the medium, the temperature, the induction time and the inducer concentration. The recombinant strain was therefore grown either on LB or 2YT medium. After induction with different concentrations of IPTG and culture during various times at room temperature, we recovered the crude extract as well as the P and the S fractions from each condition. The heterologous expression of proteins was assessed by SDS–PAGE and by dosage of the pectate lyase activity. On LB medium, after 4 h of induction, 0.4 mM IPTG was insufficient to reveal the recombinant protein while increasing the concentration of IPTG to 1.6 mM, a protein with a molecular weight of 36 kDa was observed. On 2YT medium, the hetrologous expression was improved at two levels: the concentration of 0.4 mM IPTG was sufficient and the induction time could be reduced to 2 h. This
Fig. 4. (A) SDS–PAGE gel electrophoresis (12%) of 20 g of intracellular proteins; with C: cell extract of the control strain carrying the empty vector; R: cell extract of the recombinant strain. After centrifugation, the intracellular proteins were divided in two fractions: S: supernatant representing the soluble proteins and P: pellet representing the inclusion bodies. The cultures of recombinant strains were performed on 2YT medium and the induction was carried out at 1 mM of IPTG for 3 h. (B) Profile comparison between intracellular proteins of 2YT and LB biomass, at two culture temperatures (RT and 37 ◦ C), migrated on SDS–PAGE gel electrophoresis (12%) of 20 g of proteins; with S: supernatant representing the soluble proteins and P: pellet representing the inclusion bodies.
behavior is different from the Pecate lyase of Aspergillus nidulans which was expressed in E. coli after 60 h of induction in presence of 0.5 mM IPTG [25]. Other works have reported on the great influence of yeast extract on the expression of recombinant proteins in E. coli [10]. Finally, in order to test the effect of temperature on the production level and to verify once again the difference between both media, we compared the expression on LB and 2YT at room temperature RT (about 25 ◦ C) and 37 ◦ C, while maintaining constants all the other parameters: 0.4 mM of IPTG, 150 rpm of erlenmeyers agitation and 3 h post induction. Fig. 4B shows the analysis of all S and P fractions from LB and 2YT culture conditions (RT and 37 ◦ C), Table 1 Comparison of the effects of culture conditions (the medium composition and the temperature) on the production of recombinant pectate lyase activity. LB
Tot Prot (mg/ml) Activity (units/ml)
2YT
37 ◦ C
RT
37 ◦ C
RT
3.96 4.5
4.24 14.42
5.36 7.43
7.99 41.26
Tot prot: intracellular proteins.
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Table 2 Effect of salt concentration in the binding buffer on protein recovery. 500 mM NaCl
PAL before dialysis (units) PAL after dialysis (units)
250 mM NaCl
300 mM imidazole
500 mM imidazole
1M imidazole
300 mM imidazole
500 mM imidazole
1M imidazole
5.4 7.3
0.5 3
0.1 0.2
4.1 9.2
0.2 0.3
0.2 0.3
where we can see less amounts of inclusion bodies in 2YT than in LB medium, on both culture temperature. In parallel, higher amounts of activity were recorded in the S fractions of 2YT than those of LB conditions with a clear advantage in favor of RT than 37 ◦ C culture condition, see Table 1. Therefore, all together, these results suggest that the recombinant enzyme was much more produced in soluble form than IB in 2YT than in LB and at RT than at 37 ◦ C. 3.5. Purification of the recombinant pectate lyase The optimized conditions were applied to purify the recombinant enzyme. 1 l of 2YT medium was inoculated by the recombinant strain, cultured on 37 ◦ C until reaching 0.6 as OD600 nm , then induced by 0.4 mM IPTG and incubated at room temperature with shaking at 150 rpm for 2 h. The intracellular extract was centrifuged at 13,000 × g and the supernatant was applied on MagneHis Niparticles. The binding of the His-tagged pectate lyase was firstly performed using two salt buffer (500 mM and 250 mM NaCl), then washed with 50 mM imidazole. The elution was therefore performed stepwise using increasing concentration of imidazole (50, 200 300 and 500 mM) in the two corresponding NaCl concentrations (Table 2). Interestingly, using 250 mM NaCl allowed us to elute the maximum of recombinant enzyme (9.2 units recovered
after dilaysis) at 300 mM imidazole while such concentration of imidazole was insufficient to elute all the activity when using 500 mM NaCl (only 7.3 units recovered after dialysis). Indeed, a second 500 mM imidazole step was needed to elute the remaining 3 units, compared to 0.3 units in the 250 mM condition. Moreover, even if the dialysis improved the recovery of the enzyme, it is worth to note that the recombinant enzyme was active in presence of 0.5 M NaCl and 300 mM imidazole, see Table 2, first column. Besides dosages, the electrophoresis analysis of the eluted fractions (from both 500 and 250 mM NaCl conditions) revealed a contamination of the recombinant protein with several E. coli proteins. This problem was resolved by lowering the NaCl concentration to either 100 or 0 mM. Indeed, after binding to the column, the elution was carried using various imidazole concentrations. In both conditions, the recombinant protein was eluted using only 200 mM imidazole. Fig. 5 shows the result obtained using 100 mM NaCl in the binding and elution buffer. A single and pure protein was eluted with 200 mM imidazole, at the right size of 36 kDa (Fig. 5). The recovered activity was of about 46 units, from a total of 504 units in the S fraction (see above), which means a recovery yield of slightly less than 10%. Finally, the purified and dialyzed fraction was tested for thermoactivity. The recombinant protein retained the same behavior with regard to temperature, being optimal at 60 ◦ C. Indeed, it exhibited the following activities: 25, 38 and 4 units/ml for the temperatures 50, 60 and 70 ◦ C. 4. Conclusion The cDNA of pectate lyase of P. occitanis was cloned from a cDNA bank. This cDNA was sub-cloned in the vector pET28a(+). In order to optimize the conditions for producing the pectate lyase of P. occitanis in E. coli several parameters were varied including the medium composition, the temperature, the induction time and the inducer concentration. Interestingly, we showed that the medium composition and the culture temperature greatly influence the mode of expression of our protein: it was much more expressed in soluble form on 2YT than on LB and at room temperature (about 25 ◦ C) than at 37 ◦ C. The IMAC purification method was optimized by varying the NaCl concentration during the binding and elution steps. 100 mM NaCl was the most convenient as it leads to the purification of a pure protein. This recombinant enzyme retained its thermo-active character since its optimum of activity was reached at 60 ◦ C. References
Fig. 5. Purification of the recombinant pectate lyase from E. coli. (A) Using 100 mM NaCl in binding and elution buffer. 1: eluted fractions (with 200 mM imidazole) were separated on a 12% SDS–polyacrylamide gel and stained by Coomassie blue. The arrow indicates the recombinant Pel1. M: protein markers.
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