Refolding and purification of the human secreted group IID phospholipase A2 expressed as inclusion bodies in Escherichia coli

Protein Expression and Purification 67 (2009) 82–87

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Refolding and purification of the human secreted group IID phospholipase A2 expressed as inclusion bodies in Escherichia coli Raquel Gomes Fonseca a, Tatiana Lopes Ferreira b, Richard J. Ward c,* a

Department of Biochemistry and Immunology, FMRP-USP, Universidade de São Paulo, Brazil Verdartis Desenvolvimento Biotecnológico S/S Ltda, Campus USP, Ribeirão Preto, Brazil c Department of Chemistry, FFCLRP-USP, Universidade de São Paulo, Brazil b

a r t i c l e

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Article history: Received 16 January 2009 and in revised form 3 April 2009 Available online 17 April 2009 Keywords: hsPLA2-IID Inclusion body Refolding Phospholipid hydrolysis

a b s t r a c t The secreted phospholipases A2 (sPLA2s) are water-soluble enzymes that bind to the surface of both artificial and biological lipid bilayers and hydrolyze the membrane phospholipids. The tissue expression pattern of the human group IID secretory phospholipase A2 (hsPLA2-IID) suggests that the enzyme is involved in the regulation of the immune and inflammatory responses. With an aim to establish an expression system for the hsPLA2-IID in Escherichia coli, the DNA-coding sequence for hsPLA2-IID was subcloned into the vector pET3a, and expressed as inclusion bodies in E. coli (BL21). A protocol has been developed to refold the recombinant protein in the presence of guanidinium hydrochloride, using a sizeexclusion chromatography matrix followed by dilution and dialysis to remove the excess denaturant. After purification by cation-exchange chromatography, far ultraviolet circular dichroism spectra of the recombinant hsPLA2-IID indicated protein secondary structure content similar to the homologous human group IIA secretory phospholipase A2. The refolded recombinant hsPLA2-IID demonstrated Ca2+-dependent hydrolytic activity, as measuring the release free fatty acid from phospholipid liposomes. This protein expression and purification system may be useful for site-directed mutagenesis experiments of the hsPLA2-IID which will advance our understanding of the structure–function relationship and biological effects of the protein. Ó 2009 Elsevier Inc. All rights reserved.

Introduction Phospholipases A2 (PLA2, EC 3.1.1.4)1 comprise a ubiquitous and diverse family of lipolytic enzymes that hydrolyze the sn-2 fatty acid ester bond of glycerophospholipids to produce free fatty acid and lysophospholipids [1,2]. PLA2s participate in a wide variety of physiological processes, including phospholipid metabolism, remodeling of cell membranes, and host defense [3]. These enzymes also mediate pathophysiological processes by producing precursors of biologically active lipid mediators, such as prostaglandins, leukotrienes, thromboxanes, and platelet-activating factor [3]. The PLA2s are currently classified in 15 groups on the basis of disulfide bonding patterns and amino-acid sequence similarity [4], which are * Corresponding author. Address: Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900, Monte Alegre, CEP 14040-901, Ribeirão Preto-SP, Brazil. Fax: +55 (0)16 36024838. E-mail addresses: [email protected], [email protected] (R.J. Ward). 1 hsPLA2-IID, human secreted group IID phospholipase A2; hsPLA2-IIA, human secreted group IIA phospholipase A2; IPTG, isopropyl-1-thio-b-D-galactopyranoside; NTSB, 2-nitro-5-thiobenzoic acid; UVCD, ultraviolet circular dichrosim; ADIFAB, acrylodan conjugated intestinal fatty acid binding protein; DOPC, dioleoyl phosphatidylcholine; DOPG, dioleoyl phosphatidylglycerol; SEC, size exclusion chromatography. 1046-5928/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2009.04.006

distributed between the four major classes: low molecular weight secretory PLA2 (sPLA2), cytosolic PLA2 (cPLA2), Ca2+-independent PLA2 (iPLA2) and platelet-activating factor acetylhydrolase [5]. The sPLA2 family comprises Ca2+-dependent, disulfide-rich and low molecular mass (14–18 kDa) enzymes with histidine residue in the catalytic center and a broad specificity for phospholipids with different polar head groups and fatty acyl chains [6]. To date, 10 sPLA2 isozymes (IB, IIA, IIC, IID, IIE, IIF, III, V, X, and XII) have been identified in mammals [5,7], all of which present homologous Ca2+-binding loops and catalytic sites. Awareness of the importance of sPLA2 in inflammatory responses stems from the discovery that the expression level of group IIA sPLA2 is enhanced under various inflammatory diseases [8,9], and accumulating evidence now suggests that other sPLA2 isoforms play a pivotal role in place of, or in concert with, the group IIA sPLA2 [4,10,11]. For example, antisense RNA knockdown experiments in P388D1 murine macrophages and bone marrow derived mast cells have demonstrated that the group V sPLA2s [12] are involved in the production of fatty acid precursors of inflammatory mediators [13–15]. Furthermore, the involvement of group X sPLA2 in inflammatory responses is suggested by its expression pattern that is restricted to tissues of the immune systems such as the spleen and thymus. The sPLA2-IID was first iden-

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tified in C57BL/6J mice, in which the group IIA PLA2 was naturally inactivated and yet showed a similar response to immune challenge to group IIA sPLA2-expressing mouse strains [16,17]. It was observed that the human hsPLA2-IID is expressed in the spleen and thymus [11], which is similar to the expression profile of group X sPLA2 [11,15], yet contrasts that of the group IIA hsPLA2 suggesting distinct biological functions for these related sPLA2s [11]. Although many recent studies with respect to the pharmacological effects and catalytic mechanism of recombinant hsPLA2-IIA have been reported [4,18], the human group IID sPLA2 has received little attention. This is the consequence of the technical challenges that are encountered during the refolding of the expressed protein [19], and with the exception of the hsPLA2-IID, all other human secreted PLA2s have been expressed and refolded [19]. In order to advance the characterization of the enzymatic, biological and pharmacological properties of the hsPLA2-IID, here we report the expression of the protein in the form of inclusion bodies in Escherichia coli, and the development of a refolding protocol which involves solubilization of the inclusion bodies in concentrated denaturant solutions and subsequent dilution and dialysis in order to obtain the native, and catalytically active enzyme. Materials and methods Plasmid construction A complete cDNA encoding the hsPLA2-IID (Genbank/EMBL Accession NM-012400) [11] was purchased from the IMAGE Consortium/MGC (IMAGE ID 5223912, Geneservice Ltd., Cambridge, UK), and cloned into the vector pT7T3. Restriction sites for NdeI and BamHI at the 50 and 30 ends of the protein coding region, together with a stop codon at the 3’ end, were introduced by amplification with the oligonucleotides 50 -CCAATCCATATGGCGATCCTG30 (sense) and 50 -AGGTGGGGATCCCAGAGCT CC-30 (antisense). The PCR product was separated by an agarose gel, and after digestion with NdeI and BamHI, the amplified fragment was subcloned into the equivalent restriction sites of the expression vector pET-3a. The subcloning introduced an N-terminal methionine extension into the translated protein sequence, and nucleotide sequencing confirmed the desired final construct, denoted pET3a + hsPLA2IID, in which Gly1 of the hsPLA2-IID is preceded by a Met, and a stop codon immediately follows Gly124. Expression of hsPLA2 -IID Four hundred milliliters of growth medium (2.5 g yeast extract; 10 mM MgSO4, pH 7.5; 15 lg/mL chloramphenicol; 150 lg/mL ampicillin) was inoculated with E. coli strain BL21(DE3)[pLysS] transformed with pET3a + hsPLA2-IID and grown at 37 °C to an A600 of 0.6. Recombinant protein expression was induced by addition of 0.6 mM IPTG (isopropyl-1-thio-b-D-galactopyranoside), and the culture was grown for an additional 5 h, after which time the expression of the desired product was confirmed by SDS–PAGE analysis. Inclusion bodies were isolated from bacterial pellets by repeated rounds of sonication in 40 ml of lysis buffer (50 mM Tris–HCl, pH 8.0; 1 mM EDTA; 1 mM NaCl; 0.5 mM PMSF; 0.4 M deoxycholate; 1% Triton X-100) followed by centrifugation at 12,000g. The washed inclusion bodies were divided into 6 aliquots and stored at 20 °C until used for the subsequent solubilization and refolding steps. Solubilization of inclusion bodies and refolding Each individual inclusion body aliquot (corresponding to approximately 100 mg wet weight of total protein) was solubilized

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in 0.6 mL of denaturation buffer containing sodium sulfite and 2nitro-5-thiobenzoic acid (4 M guanidinium thiocyanate, 2 mM EDTA, 300 mM Na2SO3, 50 mM NTSB) [20]. The solubilized inclusion bodies were incubated for 60 min at 45 °C to ensure complete reduction and sulfonation of cysteine residues. The mixture was subsequently passed through a BioGel P6 column (2  3 cm, BioRad) previously equilibrated in buffer (2 M guanidinium hydrochloride; 1 mM EDTA; 50 mM Tris–HCl, pH 8.0) to separate the modified protein from excess NTSB and to exchange the buffer. The protein peak was eluted in a total volume of 1.2 mL, which contained 1.2 mg/mL of total protein, and served as the starting material for the refolding process. The effects of 1–2 M argenine, argenine/guanidinium hydrochloride mixtures and choline on the refolding process were evaluated by the substitution of guanidinium hydrochloride by these reagents in the column equilibration buffer. The composition of the solutions of sulfonated protein were altered by adding 120 lL of a concentrated stock solution of an oxidation-buffer (80 mM cysteine, 10 mM cystine, 100 mM CaCl2, 50 mM Tris–HCl, pH 8.0), and incubated for 24 h at 37 °C. After incubation, a light flocculent protein precipitate formed, which was cleared by centrifugation at 12,000g. After this step, two separate refolding methods were evaluated using either refolding in the presence of a size-exclusion chromatography (SEC) matrix or by a combination of dialysis and dilution. The refolding trials in the presence of the SEC matrix were performed essentially as previously reported [31]. Briefly, the incubated protein was applied at a flow-rate of 0.2 mL/min to a Biogel P6 column (1  6.5 cm) previously equilibrated with a refolding buffer composed of either 2 M guanidinium hydrochloride or a 1 M argenine/1 M guanidinium hydrochoride mixture in 8 mM cysteine, 1 mM cystine, 1 mM CaCl2, 50 mM Tris–HCl, pH 8.0. The system was incubated at ambient temperature (20– 25 °C) for 12 h, after which the protein was eluted with 1 column volume of equilibration buffer. This protein solution was further diluted with 2 column volumes of 8 mM cysteine, 1 mM cystine, 1 mM CaCl2, 50 mM Tris–HCl, pH 8.0, and incubated at ambient temperature for 48 h with gentle agitation. After this period, the solution was passed through a 0.45 lm filter, and after adjustment of the pH to 7.5, protein was purified by cation-exchange chromatography (described in the following section). The protocol for refolding trials by a combination of dilution and dialysis used the protein incubated in solutions containing either 2 M guanidinium hydrochloride or 1 M argenine/1 M guanidinium hydrochoride mixtures in oxidation-buffer as described above. The samples were diluted over a period of 36 h to a final chaotrope concentration of 0.5 M by addition of 3 volumes of refolding buffer (8 mM cysteine, 1 mM cystine, 1 mM CaCl2, 50 mM Tris–HCl, pH 8.0). After incubation for an additional 24 h at 37 °C, the refolded protein was dialyzed against 2 L of 50 mM Tris–HCl, pH 8.0, 1 mM CaCl2 for 24 h at 25 °C. The dialyzed solution was passed through a 0.45 lm filter, and after adjustment of the pH to 7.5, protein was purified by cation-exchange chromatography. Purification of hsPLA2 -IID The reoxidized hsPLA2-IID was adjusted to pH 7.5 with HCl and purified by single-step cation-exchange chromatography using an adaptation of a previously described method [21]. Briefly, the product dialysis was applied to a Source 15S column (1  8 cm, GE Healthcare) previously equilibrated with 5 column volumes of buffer 20 mM Tris–HCl, pH 7.5. After extensive washing, the hsPLA2IID was eluted using an exponentially increasing gradient to a final concentration of 1 M NaCl in the same buffer. The eluate was continuously monitored at 280 nm, and the eluted protein peaks were

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dialyzed against 5 mM Tris–HCl, pH 7.5 for 36 h with buffer changes every 12 h, and finally concentrated approximately 10fold by solvent evaporation under vacuum and stored at 20 °C. Circular dichroism (CD) The secondary structure content of the purified hsPLA2-IID was evaluated by far ultraviolet circular dichrosim (far UV-CD) using a Jasco 810 spectropolarimeter (JASCO Corporation, Tokyo, Japan). Protein samples at a concentration of 25 lg mL1 were prepared in 10 mM KH2PO4, pH 8.0, and spectra were measured between wavelengths of 190–250 nm using 3 mm path length quartz cuvettes and a 4 nm bandwidth at a constant temperature of 298 K. The accumulated average of six protein spectra was corrected by subtraction of the spectra of the equivalent buffer in the absence of protein. The secondary structure content of the protein was estimated from the averaged and corrected far UVCD spectrum using the K2D algorithm [22]. Phospholipase A2 hydrolytic activity assay The catalytic activity of the hsPLA2-IID was evaluated by monitoring the release of the free fatty acid hydrolysis product using a phospholipid substrate. The activity was quantified by measuring the fatty acid binding to intestinal fatty acid binding protein conjugated with the fluorescent probe acrylodan (ADIFAB – InvitrogenMolecular Probes), which results in a change in the emission spectrum of the acrylodan that is directly proportional to the amount of bound fatty acid [23]. Unilamellar liposomes composed of a 9:1 M ratio of dioleoyl phosphatidylcholine (DOPC – Sigma–Aldrich, St. Louis, MO, USA) and dioleoyl phosphatidylglycerol (DOPG – Sigma–Aldrich, St. Louis, MO, USA) were prepared by reverse phase evaporation [24] and mixed at a final concentration of 2.16 lM with 3 lg/mL of ADIFAB in reaction buffer (20 mM Hepes, 20 mM NaCl, 1 mM CaCl2). hsPLA2-IID at concentrations of either 0.2 lg/ mL or 0.1 lg/mL was pre-incubated for 15 min in the same buffer, and added to the liposome/ADIFAB mixture. The fluorescence emission of ADIFAB at 505 and 425 nm was measured with an excitation wavelength of 385 nm for a period of 30 min using an SLM 3100 spectrofluorimeter, and the specific activity of the enzyme was determined from analysis of the 505 nm/425 nm signal ratio as previously described [23]. Results and discussion After 5 h induction with IPTG, E. coli (BL21) cells transformed with the construct pET-3a + hsPLA2-IID exhibited a high level of recombinant hsPLA2-IID expression (Fig. 1). The hsPLA2-IID is expressed in the form of insoluble inclusion bodies in the bacterial cytoplasm, and the isolation of these inclusion bodies is the first step in the purification process. Although group II PLA2s have a low relative molecular mass (12–15 kDa), they contain between 12 and 16 cysteine residues [4,18], and the major challenge is to overcome technical difficulties associated with refolding the polypeptide into the native state in which the correct disulfide linkages are present. The use of NTSB covalently modifies cysteine residues by adding an anionic sulfonate group [20], and is commonly used to increase the solubility of group I/II PLA2s in the initial stages of refolding, thereby reducing the concentration of chaotrope that is necessary to maintain the protein in solution. Fig. 2 shows SDS– PAGE gels from which protein yields can be qualitatively estimated before and after refolding in the presence of different compounds at total concentrations of 2 M. Fig. 2A shows the effect of buffer composition on the solubility of the hsPLA2-IID after chemical modification by NTSB as estimated by the level of protein eluted

Fig. 1. Expression of group human IID phospholipase A2 in E. coli. Equal numbers of cells were collected by measuring the A600 of the bacterial culture, and after sonication in sample buffer were analyzed using 17% SDS–PAGE. (M) molecular weight markers (Benchmark protein ladder LMW- Amersham Pharmacia); (N) extract of cells transformed with pET3a (without insert) after 5 h of IPTG induction. Lanes (1) to (5) extracts of cells transformed with pET3a + hsPLA2-IID sampled at 1 h intervals over a period of 5 h after IPTG induction.

after separation of the sulfonated polypeptide (with a molecular mass of 14 kDa) from the excess NTSB using a BioGel P6 column. Various buffers incorporating guanidinium hydrochloride, arginine and choline were evaluated (together with mixtures of these compounds), and it was observed that the best results were obtained in solutions containing guanidinium hydrochloride, either alone or as a 1:1 mixture with arginine. On the basis of this result, all subsequent steps in the refolding process used sulfonated hsPLA2-IID solubilized in a 2 M guanidinium hydrochloride solution as the starting material. Successful protein refolding from solutions containing high chaotrope concentrations in the presence of a polar size-exclusion chromatography (SEC) resin have been previously reported for snake venom group II PLA2s [21,25], and the hsPLA2-IIA [26], and these methods provided the initial conditions for the development of the refolding protocol for the hsPLA2-IID. It has been proposed that during migration of the protein solution through a SEC column, the chaotrope concentration decreases and the protein adopts a non-native compact conformation [27,28]. It would be expected that this partially refolded protein enters the resin pores whereas the unfolded protein remains in the mobile phase volume, and this partition effect favors protein refolding over aggregation [21,28–30]. These protocols use a SEC resin pre-equilibrated with chaotrope concentrations over the range 0.5–1 M, and have been successfully applied to refold group I/II PLA2s at high protein concentrations [21,26]. The influence of the SEC resin on the refolding of the hsPLA2-IID was evaluated for both the polyacrylamide-based BioGel and the agarose-based Sepharose resins, and as presented in Fig. 2B, the levels of protein eluting from the SEC resins was below the detection limit by Coomassie blue staining of SDS–PAGE gels. We conclude that in the case of hsPLA2-IID immobilization of unfolded and partially folded protein by the SEC resins used in this study do not favor protein refolding, and that exposure to the gel matrix results in tight binding with a drastic reduction in protein yield, and therefore the development of methods based on refolding in the presence of SEC resins were not pursued further. Dialysis based protocols have been successfully used to refold many group II PLA2s [19], and a simple and economical method based on step-wise dilution and a final dialysis step was developed for refolding recombinant hsPLA2-IID expressed as inclusion bodies in E. coli. After a survey of the conditions reported in the literature for the refolding of other group I/II PLA2s, guanidinium hydrochloride was chosen as the chaotrope for both the dilution and dialysis. Although only light precipitation of the protein was observed in

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Fig. 2. SDS–PAGE analysis of recombinant hsPLA2-IID refolding under various conditions. (A) Separation of the sulfonated protein from excess NTSB using BioGel P6 equilibrated with buffer containing either guanidinium hydrochloride (2 M) (Gnd), arginina (2 M) (Arg), guanidinium hydrochloride (1 M) plus arginine (1 M) (Gnd + Arg), choline (2 M) (Cho), guanidinium hydrochloride (1 M) plus choline (1 M) (Gdn + Cho). (B) Refolding of the sulfonated protein in the presence of size-exclusion chromatography (SEC) resins equilibrated with buffer containing either guanidinium hydrochloride (2 M) (Gnd) or guanidinium hydrochloride (1 M) plus arginine (1 M) (Gnd + Arg). Lane B, sample of hsPLA2-IID after refolding in the presence of BioGel P6 resin; Lane S, sample of hsPLA2-IID after refolding in the presence of Sepharose resin. (C) Refolding by dialysis in buffer containing either guanidinium hydrochloride (Gnd), or guanidinium hydrochloride (1 M) plus arginine (1 M) (Gnd + Arg). See materials and methods section for details. Lane MP, marker proteins showing corresponding molecular weights.

the dilution steps, heavy precipitation occurred during final dialysis, reducing the final yield of the overall process. Fig. 2C shows an SDS–PAGE gel of the soluble protein fraction after dialysis, and attempts to improve the final yield of native protein using mixtures of arginine or arginine were not successful. The best yields were obtained from a protocol combining 12 h incubation in 2 M guanidinium hydrochloride in oxidation buffer, followed by three consecutive dilution steps over a 36 h period, with a final dialysis to remove the remaining guanidinium hydrochloride (see materials and methods for details). The reoxidation product resulting from the final dialysis step was purified by cation-exchange chromatography, and as shown in Fig. 3 the protein peak (indicated as peak P2 in the chromatogram) eluted at between 8 and 9 min. Coomassie blue stained SDS–PAGE analysis of this fraction demonstrates that the single

Fig. 3. Elution profile of the refolded hsPLA2-IID protein using cationic exchange chromatography. The column was previously equilibrated in 20 mM Tris-Cl pH 7,5 and eluted with a gradient of 20 mM Tris-Cl, 1 M NaCl pH 7.5. Peak 2 contained the correctly refolded protein as verified by the far ultraviolet circular dichrosim spectrum, and hydrolytic activity assays. The insert shows the SDS–PAGE analysis of recombinant hsPLA2-IID purified by single step cation-exchange chromatography. Lane P2; protein peak 2 from the recombinant hsPLA2-IID purification, Lane M; molecular weight marker.

step ion-exchange purification method resulted in protein purity greater than 99% (insert to Fig. 3), and the complete refolding and purification process typically yields in a final yield of 35– 40 lg of protein per liter of bacterial culture. Fig. 3 shows that the chromatogram presents a minor peak that appears at approximately 7 min, and blank gradient runs in the absence of protein showed that this peak is due to absorption artifacts probably arising from the changing salt concentration. The secondary structure content of the protein eluted in the major fraction (peak P2) was estimated by circular dichrosim (CD) analysis. The profile of the far UV CD spectrum (Fig. 4) shows minima at 208 and 222 and a maximum at 194 nm, which is typical of a protein with a high a-helical content, and secondary structure content prediction using the K2D algorithm [22] yielded values of 45% a-helix and 11% b-strand. The amino-acid sequence of the hsPLA2-IID and the hsPLA2-IIA share 50.4% identity, suggesting that the two proteins are homologous, and it would be expected that they possess similar secondary structure composition. The crystal structure of the hsPLA2-IIA [27] shows 42% a-helix and 8% b-strand, which correlates well with the secondary structure content estimated by far UV-CD analysis of the hsPLA2-IID.

Fig. 4. Far ultraviolet circular dichrosim spectra of the recombinant hsPLA2-IID.

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Kinetic measurements of phospholipid hydrolysis were quantified by measuring fatty acid released from DOPC/DOPG liposomes on incubation with recombinant hsPLA2-IID. Fig. 5 presents protein concentration dependence of substrate hydrolysis, and demonstrates that the catalytic activity of the hsPLA2-IID involves a Ca2+-dependent mechanism. Amino-acid sequence comparison of the hsPLA2-IID with other group II PLA2s shows that the residues Cys29GlyXGlyGly33 and Asp49 that are involved in binding the calcium co-factor are fully conserved [11]. Indeed, the dependence of the Ca2+co-factor was demonstrated by the loss of hydrolytic activity of the hsPLA2-IID in the presence of EGTA (see Fig. 5). Substrate hydrolysis in group I/II sPLA2s proceeds through the activation and orientation of a water molecule by hydrogen bonding to the active site His48, and the active site residues Asp42XCysCysXXHisAsp49 are also conserved in the hsPLA2-IID [11]. The specific activity of phospholipid hydrolysis by the recombinant hsPLA2-IID against the mixed DOPC/DOPG substrate was 63 lmol min1 mg1, which is low in relation to the hsPLA2s-IIA. It is noteworthy that recombinant murine sPLA2-IID also demonstrates a significantly lower activity against DOPG substrates when compared to the murine sPLA2-IIA [11]. This reduced activity may be due to differences in the affinity for the substrate between the sPLA2-IID and sPLA2sIIA, which might be result of amino-acid substitutions in the substrate binding region of the protein. In the hsPLA2s-IID, the highly conserved F5/A102/F106 (FAF) triad that is observed in the hydrophobic substrate-binding cleft of group II PLA2s is substituted by the L5/V102/L106 (LVL) triad. It is noteworthy that the LVL triad is also observed in the Group II Lys49-PLA2s, and structural comparison with other group II PLA2s reveals that the FAF to LVL substitution maintains the hydrophobic packing volume of the residues lining the wall of the substrate-binding cleft, yet alters the topology of this region [31]. Substitution of the LVL triad by FAF in a Lys49-PLA2s resulted in a reduction in the membrane permeabilizing activity of the protein, which was interpreted as the consequence of an alteration in the lipid binding affinity [32]. To the best of our knowledge, the present study represents first published report of the successful heterologous expression, refolding and purification of catalytically active recombinant human sPLA2-IID. The protocol described here uses a simple and inexpensive combination of dilution and dialysis, which facilitates the simultaneous processing of multiple recombinant protein samples.

Fig. 5. Hydrolytic activity determination of the recombinant hsPLA2-IID. The enzymatic activity was determined by measuring the change in the ADIFAB fluorescence ratio at 505 nm in relation to 425 nm (R505/425). (A) 0.2 lg mL1 hsPLA2-IID + 2 mM EGTA, (B) 0.1 lg mL1 hsPLA2-IID + 1 mM Ca2+, (C) 0.2 lg mL1 hsPLA2-IID + Ca2+. In all measurements the lipid concentration was 2.16 lM, and the ADIFAB concentration was 3 lg mL1. In each experiment, the hsPLA2-IID was added approximately 500 s after the start of the scan. Specific sPLA2 activity was determined as described in the materials and methods section.

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