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Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep 5 6

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Expression and purification of biologically active recombinant human paraoxonase 1 from inclusion bodies of Escherichia coli

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Priyanka Bajaj, Rajan K. Tripathy, Geetika Aggarwal, Abhay H. Pande ⇑

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Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar (Mohali) 160062, Punjab, India

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Article history: Received 3 April 2015 and in revised form 13 May 2015 Available online xxxx Keywords: Acyl homoserine lactone Homocysteinethiolactone In vitro refolding Inclusion bodies Organophosphate

a b s t r a c t Human PON1 (h-PON1) is a Ca2+-dependent serum enzyme and can hydrolyze (and inactivate) a wide range of substrates. It is a multifaceted enzyme and exhibit anti-inflammatory, anti-oxidative, anti-atherogenic, anti-diabetic, anti-microbial, and organophosphate (OP)-detoxifying properties. Thus, h-PON1 is a strong candidate for the development of therapeutic intervention against these conditions in humans. Insufficient hydrolyzing activity of native h-PON1 against desirable substrate affirms the urgent need to develop improved variant(s) of h-PON1 having enhanced activity. Production of recombinant h-PON1 (rh-PON1) using an Escherichia coli expression system is a key to develop such variant(s). However, generation of rh-PON1 using E. coli expression system has been elusive until now because of the aggregation of over-expressed rh-PON1 protein in inactive form as inclusion bodies (IBs) in the bacterial cells. In this study, we have over-expressed rh-PON1(wt) and rh-PON1(H115W;R192K) proteins as IBs in E. coli, and refolded the inactive enzymes present in the IBs to their active form using in vitro refolding. The active enzymes were isolated from the refolding mixture by ion-exchange chromatography. The catalytic properties of the refolded enzymes were similar to their soluble counterparts. Our results show that the pure and the active variant of rh-PON1 enzyme having enhanced hydrolyzing activity can be produced in large quantities using E. coli expression system. This method can be used for the industrial scale production of rh-PON1 enzymes and will aid in developing h-PON1 as a therapeutic candidate. Ó 2015 Published by Elsevier Inc.

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Introduction

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Human paraoxonase 1 (h-PON1)1 (EC 3.1.8.1) is a 45 kDa, 2+ Ca -dependent serum enzyme that can hydrolyze a variety of substrates [1,2]. It is primarily synthesized in the liver and is secreted into the bloodstream where it is associated with a category of high density lipoprotein particles [3,4]. The h-PON1 is a multitasking enzyme and various hydrolytic activities of h-PON1 can be grouped into three categories: arylesterase, phosphotriesterase and lactonase [5]. The precise physiological function(s) of h-PON1 is not known yet; however, the enzyme has shown to exhibit anti-inflammatory, anti-oxidative, anti-atherogenic, anti-diabetic, anti-inflammatory and organophosphate (OP)-hydrolyzing properties [6–13]. Recent reports suggest that h-PON1 also plays an important role in the metabolism of certain drugs [14,15].

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⇑ Corresponding author. Tel.: +91 172 2214 682; fax: +91 172 2214 692. E-mail addresses: [email protected], [email protected] (A.H. Pande). 1 Abbreviations used: AHL, acyl homoserine lactone; CPO, chlorpyrifos oxon; CWNA, chemical warfare nerve agent; h-PON1, human paraoxonase 1; rh-PON1, recombinant human paraoxonase 1; OP, organophosphate; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

The level and the activity of serum PON1 in individuals suffering from cardiovascular diseases, liver diseases, diabetes, renal diseases, cancer, and obesity are considerably lower than in the normal subjects [6,16,17]. Animals deficient in PON1 have been found to be more susceptible to these disease conditions and the over expression of h-PON1 or administration of purified PON1 in these animals has been shown to prevent/retard the development of these disease conditions [7–10]. Thus, h-PON1 is a strong candidate for the development of therapeutic intervention against these disease conditions in humans. The beneficial role of h-PON1 in OP-poisoning is also well demonstrated. OP-compounds are toxic chemicals that exert their harmful effect by inhibiting the function of neurotransmitter-metabolizing enzymes [18]. OP-compounds are easy to manufacture and are widely used as pesticides, fungicides, insecticides, herbicides and petroleum additives in agriculture and other industries. Certain OP-compounds developed by the armies as chemical warfare nerve agents (CWNAs) are much more dangerous and have become important terrorist chemical weapon in today’s world [18,19]. Animals deficient in PON1 have been found to be more susceptible to OP-poisoning and the over-expression of h-PON1 or administration of purified h-PON1 into transgenic animals has been shown to

http://dx.doi.org/10.1016/j.pep.2015.05.011 1046-5928/Ó 2015 Published by Elsevier Inc.

Please cite this article in press as: P. Bajaj et al., Expression and purification of biologically active recombinant human paraoxonase 1 from inclusion bodies of Escherichia coli, Protein Expr. Purif. (2015), http://dx.doi.org/10.1016/j.pep.2015.05.011

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prevent/retard their susceptibility to OP-poisoning [20–24]. In humans, the amount and the level of activity of serum PON1 have been demonstrated to have an impact on determining their susceptibility towards OP-poisoning [25–27]. Thus, h-PON1 is a strong candidate for the development of antidote against OP-poisoning in humans. Native h-PON1 does not have sufficient catalytic activity against all of its substrates; therefore, there is a need to develop improved variant(s) of h-PON1 having enhanced activity against desired substrate(s). Production of recombinant h-PON1 (rh-PON1) using Escherichia coli expression system, a most preferred system for the manufacture of recombinant proteins, is a key to develop better variants of h-PON1. However, various attempts to generate active rh-PON1 with high purity and high yield using this expression system were unsuccessful [28–31]. Being a eukaryotic protein, over-expression of rh-PON1 in E. coli leads to aggregation of recombinant protein in inactive form as inclusion bodies (IBs), making it difficult to produce rh-PON1 enzymes in active form with high yield using E. coli expression system [28–31]. Production of active recombinant proteins by in vitro refolding of inactive proteins present in IBs have emerged as an attractive alternative method over soluble production of these recombinant proteins [32–34]. However, the process of refolding of inactive proteins present in IBs to their active form is recognized to cause a major bottleneck in the protein production scheme and method(s); hence, refolding of recombinant proteins should be developed on a case-by-case basis [33,34]. In this study, we have over-expressed rh-PON1(wt) and rh-PON1(H115W;R192K) proteins as IBs in E. coli and refolded the inactive enzymes present in the IBs to their active form using in vitro refolding. The active enzymes were isolated from the refolding mixture by ion-exchange chromatography. The catalytic properties of the refolded enzymes were similar to their soluble counterpart.

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Materials and methods

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Materials

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5-bromo-4-chloro-3 -indolyphosphate (BCIP), nitro-bluetetrazolium (NBT) reagent, alkaline phosphatase-labeled anti-mouse secondary antibody, arginine, CHES buffer, dextrose, guanidine HCl, Hepes buffer, isopropyl-1-b-D-galactopyranoside (IPTG), lactose, lysine, lysozyme, maltose, mannitol, mannose, m-cresol purple, methyl b-cyclodextrin, MOPS buffer, NDSB201, NDSB256, oxidized glutathione, paraoxon ethyl, PIPES buffer, polyethylene glycol (PEG)-400, -1000 and -3350, proline, protease inhibitor cocktail, reduced glutathione, sorbitol, TAPS buffer, Tris (2-carboxyethyl) phosphine (TCEP), trehalose, Tris–HCl, Triton X-100, Tween-20, Tween-80 and urea were purchased from Sigma–Aldrich, Bangalore, India. E. coli BL21 (DE3), DH5a cells and pET23a(+) plasmid were purchased from Merck (Novagen) Bangalore, India. Gene JET™ gel extraction kit, T4 DNA ligase, dNTPs, restriction enzymes, nuclease free water and DNA ladder were obtained Fermentas, CA, USA. Protein molecular weight markers and Bradford reagent were purchased from Bio-Rad, Gurgaon, India. Q-Sepharose columns were from GE Healthcare (GE Healthcare Bio-Sciences Ltd., Uppsala, Sweden). All other reagents used were of highest analytical grade. Construction of expression plasmid containing gene for rh-PON1 enzyme Construction of expression plasmids containing codon optimized gene encoding soluble rh-PON1 enzymes is described elsewhere [30,31]. These recombinant enzymes contained 355

amino acids of native h-PON1 along with one extra amino acid (glutamic acid) at 356th position and a (His)6-tag. The soluble recombinant proteins were ‘humanized’ by removing the extra amino acid, as well as a C-terminal (His)6-tag via a PCR amplification reaction. For this, plasmids containing gene encoding rh-PON1 enzymes were purified and amplified in a PCR reaction using following primers; F-(50 -GGAATTCCATATGGCGAAACTGATTGCCCT G-30 ) and R-(50 -CCGCTCGAGTCAGAGTTCGCAATACAGCGCTTT-30 ). The forward primer contain Nde1 restriction site and the reverse primer contain a stop codon followed by a Xho1 restriction site. The amplified genes and empty pET23a(+) plasmid (1 lg each) were separately subjected to restriction digestion by mixing with 1 U of Nde1/Xho1 restriction enzymes and 1  of appropriate digestion buffers in a final volume of 20 ll. All the digestion reactions were carried out for 12 h at 37 °C and the digested DNA products were resolved on (1%) agarose gel electrophoresis. The linearized plasmid backbone and open reading frame (ORF) dropouts were purified from the agarose gel using Gene JET™ gel extraction kit and ligated using T4 DNA ligase (plasmid backbone and ORF ratio of 1:3) to generate pET23a(+) plasmids containing gene for ‘humanized’ rh-PON1 enzymes. The ligation reaction was incubated at 16 °C for 16 h and transformed into E. coli DH5a cells. The presence of desirable ORF in the transformed plasmids was confirmed by restriction digestion of the purified plasmids with Nde1/Xho1 restriction enzymes, as well as by direct DNA sequencing (Eurofinn, Bangalore, India). The plasmid containing rh-PON1 enzymes were then transformed into E. coli BL21(DE3) cells and glycerol stocks of the transformed cells were prepared and stored at 80 °C.

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Expression of rh-PON1 and purification of IBs

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Glycerol stock of E. coli BL21(DE3) cells containing pET23a(+)-rh-PON1 was streaked on a LB-agar plate containing 50 lg/ml carbenicillin and 1 mM CaCl2 and incubated overnight at 37 °C. A single colony from the plate was picked and inoculated into LB-broth supplemented with 50 lg/ml carbenicillin and 1 mM CaCl2 and was grown at 37 °C overnight (seed culture). Of the seed culture, 1% was withdrawn and then inoculated into fresh LB-broth supplemented with 50 lg/ml carbenicillin and 1 mM CaCl2 and the main culture was grown at 37 °C until OD600 reached 0.6–0.8. The culture was then induced with 1 mM IPTG and the cells were further allowed to grow at 37 °C for 8 h. The cells were harvested by centrifugation (3–4g wet cell mass per liter of culture) and were used to purify IBs containing rh-PON1 enzyme, by following a procedure described in ref [32,35], with slight modification. Briefly, the cell pellet was re-suspended in ice-cold lysis buffer (50 mM Tris–HCl, pH 8.0 containing 150 mM NaCl, 1 mM CaCl2, 1 mM b-ME, 0.1 mM of protease inhibitor cocktail and 10 lg/ml lysozyme) at a ratio of 1:10 (w/v). The cell suspensions were subjected to sonication. The sonicated cell suspensions were immediately cooled on ice and treated with DNase (10 lg/ml) and 0.3 mM MgCl2 for 1 h. Then 2 V of buffer (100 mM Tris–HCl, pH 7.0 and containing 2% Triton X-100, 1.5 M NaCl and 20 mM EDTA) were added to the sample to make the total volume 3 V. The samples were centrifuged (10,000g, 30 min, 4 °C) to separate clear cell lysates from insoluble fraction containing rh-PON1-enriched IBs. The IBs were then washed twice with 400 ml of IB-washing buffer (100 mM Tris–HCl, pH 7.0 containing 20 mM EDTA) and centrifuged (10,000g, 30 min, 4 °C) to remove the contaminants present in the IBs. Purified IBs were then collected and stored at 80 °C until further use. Fractions containing proteins were analyzed by SDS–PAGE and western blot analysis. Monoclonal mouse anti-human PON1 antibody was used as a primary antibody in the western blot analysis.

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In vitro refolding

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To identify the buffer condition that could give maximum refolding of rh-PON1 enzymes, purified IB was re-suspended in freshly prepared 8 M urea solution (1 mg/ml) until a transparent and homogeneous solution was obtained. The sample was centrifuged (10,000g, 30 min, 4 °C) to remove the residual insoluble material and the clear supernatant containing denatured rh-PON1 protein was used for in vitro refolding. Different refolding additives that are known to facilitate in vitro refolding of recombinant proteins were selected: a buffering agent selected from the group of Tris–HCl, CHES, EPPS, HEPES, Glycine-NaOH, Phosphate, TAPS, MOPS, and MEPES; a cofactor selected from the group of CaCl2, CoCl2, MgCl2, and ZnCl2; a salt selected from the group of NaCl, KCl, and NH4Cl; a detergent selected from the group of Tween-20, NP-10, NP-40, Triton X-100, CHAPS, and Brij 35; an amino acid selected from the group of arginine, lysine, histidine, glutamic acid, aspartic acid, glycine, alanine, proline, serine, threonine, tryptophan, phenylalanine, cysteine, methionine, valine, leucine, isoleucine, tyrosine, asparagine and glutamine; a sugar selected from a group of maltose, glucose, mannose, trehalose, sucrose, dextrose, lactose, glycerol, sorbitol, mannitol, myo-inositol, xylitol, and ethylene glycol; a polymer selected from a group of cyclodextrins polyethylene glycols; a surfactant selected from a group of NDSB201 and NDSB256; and a reducing agent selected from GSH, TCEP and DTT. The refolding buffers were prepared fresh in-house and used immediately to avoid possible intervention in stability of any additive. To identify the buffer composition giving maximum refolding of rh-PON1 enzymes, denatured rh-PON1 protein solution (0.5 mg/ml) was rapidly diluted (1:50) in refolding buffer (250 ll) containing various combinations of chemical additives. Since calcium is an essential cofactor for h-PON1, all refolding buffers were ensured that it contained 1 mM CaCl2. The refolding reactions were incubated at 25 °C with gentle shaking. After incubation period, the extent of refolding was checked by monitoring the paraoxonase activity of enzyme using paraoxon as substrate. Buffer containing no protein was taken as control to correct the spontaneous, non-enzymatic hydrolysis of the substrate and was subtracted from the total hydrolysis by the enzyme. The activity of same amount of purified (His)6-tagged rh-PON1 enzyme (soluble counterpart) was taken as 100%. All the refolding experiments were performed in duplicate.

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Isolation of enzymatically active rh-PON1 present in the refolding reaction Denatured solution of rh-PON1 enzyme (1 mg/ml in 8 M urea) was rapidly diluted (1:50) in refolding buffer (5 L), and the solution was stirred for 12 h at 25 °C. The refolding mixture was then applied onto a Q-Sepharose column (20 ml) pre-equilibrated with Tris buffer (20 mM Tris–HCl, pH 8.0 containing 1 mM CaCl2) containing 0.05% Tergitol. After washing the column with same buffer, the bound proteins were eluted by increasing the concentrations of NaCl (0.1–1 M) in the same buffer. Eluted fractions were analyzed for both protein contents (OD280) and enzyme activity (using paraoxon as substrate). The fractions containing proteins were pooled, concentrated using Amicon concentrator (MWCO 3 kDa) and stored at 4 °C. The fractions were analyzed using SDS–PAGE and western blot analysis. Comparison of specific activity of refolded and soluble rh-PON1 enzymes Equal amount of protein was taken in Tris buffer, and the paraoxonase activity was determined using paraoxon as a

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substrate. The final concentration of paraoxon was 1 mM and the formation of p-nitro phenol was monitored at 405 nm for a fixed period of time. Enzymatic activity was calculated from the molar extinction coefficient of p-nitro phenol (e405 = 9000 M1 cm1) and corrected for the non-enzymatic hydrolysis [29]. Specific activity was calculated and represented as micromole substrate hydrolyzed/min/mg of protein.

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Lactonase activity of refolded rh-PON1 enzymes

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Homocysteinethiolactone (HTLactone), and acylhomoserine lactone (AHL)-hydrolyzing activity of enzymes was determined as described in our previous reports [30,31]. Appropriate blanks were included to correct for the spontaneous, non-enzymatic hydrolysis of substrates and was subtracted from the total rate of hydrolysis.

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Results

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Earlier we had expressed, purified, and characterized the enzymatic properties of variants of rh-PON1 in our lab [30,31]. These rh-PON1 variants were expressed in E. coli cells as soluble and active enzymes. These recombinant enzymes contained 355 amino acids of native h-PON1 along with one extra amino acid (glutamic acid) at 356th position and a (His)6-tag. These recombinant enzymes were referred to as ‘soluble’, to differentiate them from the refolded enzymes described in this report. Being therapeutically important protein, the presence of extra amino acid or ‘tag’ in the recombinant protein of h-PON1 may lead to complications when used as a drug; therefore, the soluble recombinant proteins were ‘humanized’ by removing the extra amino acid as well as the C-terminal (His)6-tag through a PCR amplification reaction [32]. Fig. 1A shows a comparison of the nucleic acid sequence coding for ‘humanized’ rh-PON1(wt) with the nucleic acid sequence of native h-PON1 (GenBank # P27169). At nucleic acid level, the gene for rh-PON1(wt) exhibits only 37% similarity with the gene for native h-PON1. However, comparison of the deduced amino acid sequences of rh-PON1(wt) with native h-PON1 indicates that both proteins share 100% identity with each other (Fig. 1B). Various catalytic properties of rh-PON1(wt) were comparable to that of the native h-PON1 [30,31]. In this study, we have also used a variant of rh-PON1(wt) containing substitution at 115 and 192 positions (rh-PON1(H115W;R192K)).

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Expression of rh-PON1 enzyme in E. coli to form IBs and purification of IBs

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E. coli cells expressing rh-PON1 were grown in Luria–Bertani (LB) media, and the culture was induced with 1 mM IPTG for 8 h at 37 °C to over express the recombinant protein as IBs [32]. IBs were then purified from the bacterial cells, and the purity of IBs was assessed by monitoring the protein content at different stages of purification done through SDS–PAGE and western blot analysis (Fig. 2). Though small amounts of recombinant protein was always observed in the cell lysate (soluble) fraction and washes, most of the rh-PON1 protein was present in the pure form in IBs. This purified, but inactive, rh-PON1 protein was used further for in vitro refolding.

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In vitro refolding of rh-PON1 enzyme

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From the literature, it is evident that condition for optimum refolding of recombinant proteins (present in IBs) from an inactive form to their active form varies from protein to protein, and no single refolding method or buffer composition satisfy all protein

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Fig. 1. Comparison of gene (A) and deduced amino acid sequence (B) of rh-PON1(wt) and native h-PON1. Sequence alignment of both the nucleotide and amino acid sequences of rh-PON1(wt) and native h-PON1 was done by using free online software clustalW and improved manually. Dashes and shaded area represents identical and dissimilar nucleotides/amino acids, respectively.

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refolding processes [36,37]. Small chemical compounds (refolding additives) have been used in the past by other researchers to successfully refold variety of proteins [38–41]. Also, one of the simplest and economical methods of in vitro refolding of recombinant protein is to dilute the concentrated solution of denatured protein (IBs denatured in a particular chaotropic agent) into the refolding buffer containing a particular combination of refolding additives [38–41]. Purified IBs (enriched with inactive rh-PON1) (Fig. 2) were dissolved in freshly prepared 8 M urea to denature the recombinant protein. To identify the appropriate refolding buffer (buffer condition and refolding additive compositions) that can give maximum refolding of rh-PON1, 5 ll of the denatured rh-PON1 solution (0.5 mg/ml) was rapidly added to the wells of 96-well plate containing 250 ll of different refolding buffers (310 combinations),

and the protein was allowed to refold by incubating the plate at 25 °C. Refolding of rh-PON1 was checked by monitoring the paraoxonase activity of the enzyme, as described in ‘Materials and Methods’ section. The activity of the enzyme was observed in many wells suggesting that many refolding buffers can refold the rh-PON1 enzyme to its active form. One refolding buffer (200 mM TAPS, pH 8.5 containing 1.0 M NDSB 201, 1 mM EDTA, 2.2 mM GSH, 0.22 mM GSSH and 10 mM CaCl2) that gave maximum refolding of rh-PON1 enzyme was selected and used to optimize other parameters. Besides chemical compositions of the refolding buffer, number of other factor also influence the final yield of active (refolded) proteins in in vitro refolding procedure [42–44]. Among these, the most important factors are the type of chaotropic agent used to denature the recombinant proteins present in IBs, concentration

Please cite this article in press as: P. Bajaj et al., Expression and purification of biologically active recombinant human paraoxonase 1 from inclusion bodies of Escherichia coli, Protein Expr. Purif. (2015), http://dx.doi.org/10.1016/j.pep.2015.05.011

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Fig. 2. Purification of IBs of rh-PON1 enzyme. Panels A and B depict representative images of Coomassie stained SDS–PAGE (4–20% gradient) and western blot, respectively, of fractions collected during typical purification of IBs of rh-PON1 enzyme. Legends: lane M, protein molecular weight markers; lane 1, cell lysate; lane 2 and 3, supernatants of the IBs washings 1 and 2; and lane 4, purified IBs. 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396

of recombinant protein used in the refolding reaction, and the temperature and the time of refolding reaction [42–44]. Next, we studied the effect of these parameters on the extent of refolding of rh-PON1 enzyme. We observed that, among the various other conditions that were tried, the extent of refolding of rh-PON1 was more when (a) 8 M urea was used as a chaotropic agent to denature the rh-PON1 present in IBs, (b) low concentration of rh-PON1 protein (5 lg/ml final concentration) was used in the refolding reaction, and (c) when the refolding reaction was incubated for 12 h at 25 °C. Since using a low concentration of protein in in vitro refolding is generally not economical for large-scale production of protein [44], we selected the protein concentration of 20 lg/ml for the refolding reaction. The selected buffer composition and standardized conditions were used to refold rh-PON1 enzymes. Isolation of enzymatically active rh-PON1 present in the refolding reaction Various chromatographic approaches (like ion-exchange, size-exclusion or hydrophobic interaction chromatography) have been used in the literature to separate active and correctly folded proteins from their misfolded/partially folded forms present in the refolding reaction [44]. It is also known that refolded material often contains soluble multimers that bind to ion-exchange columns tighter than monomers [45]. Enzymatically active (and correctly folded) rh-PON1 was separated from its inactive (misfolded/partially folded) forms present in the refolding reaction by using ion-exchange chromatography. After in vitro refolding, the refolding reaction was loaded onto a Q-Sepharose column. The column was washed to remove unbound protein, while the bound protein was eluted by increasing the concentration of NaCl (0.1– 1 M) in the elution buffer. A typical chromatogram showing resolution of proteins is given in Fig. 3A. The protein present in the refolding reaction was eluted in three peaks: a minor peak eluted at 0.1–0.2 M NaCl followed by two major peaks (peak P1 and P2) at 0.3–0.4 M and 1 M NaCl in the elution buffer, respectively. Measurement of the paraoxonase activity in the collected fractions suggested that the protein eluted in peak (P1) was fully active while the protein in the other two peaks was enzymatically inactive. SDS–PAGE and western blot analysis of the fractions indicated that rh-PON1 protein was present in both peak fractions (as a band of 45 kDa; Fig. 3B and C). Presence of an additional (minor) band along with the major band in the samples could be due to anomalous running of proteins during electrophoresis. However, the presence of different forms of refolded and active enzyme in purified preparation cannot be ruled out. Nevertheless, our results indicate

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that ion-exchange chromatography is able to separate the active (refolded) rh-PON1 enzyme from its inactive form present in the refolding reaction. Using this purification procedure the average recovery and the percentage yield of purified rh-PON1 enzymes from isolated IBs were 28 mg of protein (per liter of E. coli culture) and 24%, respectively.

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Comparison of specific activity of refolded and soluble rh-PON1 enzymes

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Quality of the refolded proteins (especially enzymes) is measured in terms of their catalytic activity. Paraoxonase activity of the refolded rh-PON1(wt) and rh-PON1(H115W;R192K) was compared with their soluble counterpart to determine the quality of the refolded proteins (Fig. 4). The specific activity of the refolded enzymes was comparable with the specific activity of (His)6-tagged soluble counterparts [30]. This indicates that the refolded rh-PON1 enzymes are identical to their soluble counterpart in terms of catalytic activity.

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Characterization of lactonase activity of refolded rh-PON1 enzymes

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The native activity of h-PON1 is lactonase [5,46]. The lactonase activity of refolded rh-PON1 enzymes was determined using two well-known lactone substrates of h-PON1: HTLactone and AHL. HTLactone is a metabolic byproduct of methionine metabolism and is generated from homocysteinyl [47]. It is a reactive thioester that causes N-homocysteinylation of proteins in the body. The increased plasma HTLactone level is linked with increased risk of various inflammatory disorders [47]. HTLactone-hydrolyzing activity of the refolded rh-PON1 enzymes was compared by using a pH-indicator based colorimetric assay and the result is presented in Fig. 5A. Compared to rh-PON1(wt), rh-PON1(H115W;R192K) showed relatively less HTLactone-hydrolyzing activity. AHLs are used by many Gram-negative bacteria as a quorum sensor signals that enable the bacteria to modulate the expression of genes involved in biofilm formation, bacterial virulence, antibiotic production, sporulation, tissue damage, etc [48]. H-PON1 can inactivate AHLs by hydrolyzing the lactone ring and thus provide protection against bacterial infection [49]. AHL-hydrolyzing activity of refolded rh-PON1 enzymes was determined by comparing their capacity to inhibit the 3O-C12AHL-induced expression of reporter gene (b-galactosidase) in the recombinant quorum-sensing reporter E. coli strain [30]. Compared to rh-PON1(wt), rh-PON1(H115W;R192K) showed increased 3O-C12AHLhydrolyzing activity (Fig. 5B). The lactonase activity of the refolded enzymes was comparable with the activity of their soluble counterpart and depend on the type of the lactone substrate [30,31].

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Discussion

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H-PON1 can hydrolyze and inactivate variety of OP-compounds, therefore, is considered as a promising candidate for the development of therapeutic against various conditions in humans [11,12,25–27]. Since, native h-PON1 does not possess sufficient catalytic activity against number of its substrates, it is important to engineer and develop improved variant(s) of h-PON1 having enhanced hydrolytic activities against the desired substrate(s). However, production of the recombinant h-PON1 using bacterial expression system have been difficult until now, making it hard to produce improved variants of h-PON1 and analyzing their in vivo efficacy. In our lab, we have expressed variants of rh-PON1 enzyme in soluble and active form as (His)6-tagged proteins in E. coli [30,31]. However, the yield of the purified rh-PON1 enzymes was very low, despite growing the cells

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Fig. 3. Isolation of enzymatically active rh-PON1 present in the refolding reaction. Panel A represent a chromatogram illustrating the separation of active rh-PON1 enzyme from the inactive fraction by using Q-Sepharose column. (-O-) and ( ) denotes the absorbance at 280 nm and paraoxonase activity of the eluted fractions from the columns, respectively. ( ) represent increasing ionic strength (NaCl concentration) of the elution buffer. P1 and P2 represents two major peaks of proteins eluted at different concentrations of NaCl. Panels B and C depict images of Coomassie stained SDS–PAGE (4–20% gradient) and western blot, respectively, of the fractions obtained at various stages of protein separation. The western blot was developed using monoclonal mouse anti-human PON1 antibodies as primary antibody. Legends: lane M, protein molecular weight markers; lane 1, protein refolding mixture loaded onto the column; lane 2, wash; lane 3, pooled fractions from peak 1 having active rh-PON1 enzyme; and lane 4, pooled fractions from peak 2 having inactive rh-PON1 enzyme.

Fig. 4. Comparison of the OP-hydrolyzing activity of the refolded rh-PON1 enzyme. Specific activity of refolded (j) and soluble ( ) rh-PON1 enzymes were compared using paraoxon as a substrate. Equal amount of protein was taken and the paraoxonase activity was determined. The final concentration of paraoxon was 1 mM. Enzymatic activity was calculated from the molar extinction coefficient of pnitro phenol, corrected for the non-enzymatic hydrolysis, and the specific activity was calculated. p > 0.05, refolded vs soluble enzymes.

Fig. 5. Lactonase activity of the refolded rh-PON1 enzyme. (Panel A) HTLactonehydrolyzing activity of refolded rh-PON1 enzymes. Legends: bar-1, rh-PON1(wt), and bar-2, rh-PON1(H115W;R192K). (Panel B) 3O-C12AHL-hydrolyzing activity of refolded rh-PON1 enzymes. The AHL-hydrolyzing activity of enzymes was determined by using a recombinant quorum-sensing reporter E. coli strain. Legends: bar-1, control (only 3O-C12AHL and no enzyme); bar-2, rh-PON1(wt); and bar-3, rhPON1(H115W;R192K).

expressing rh-PON1 enzyme at low temperature and using low concentration of inducer (i.e., IPTG), the two most important parameters that promotes the expression of recombinant proteins in functionally active form in E. coli [43]. This is because the majority of the recombinant protein was expressed as biologically inactive aggregates in the form of IBs in E. coli cells. Also, these recombinant enzymes contained (His)6-tag that helped in the purification of recombinant proteins [30,31]. However, the presence of ‘tag’ in therapeutically important proteins may lead to complications when such proteins are used as a drug. To address these issues, we have we have refolded the inactive rh-PON1 enzymes (which do not contain (His)6-tag) present in IBs to their active form by in vitro refolding. The active enzyme in the refolding mixture was separated from its inactive form by ion-exchange chromatography. The refolded rh-PON1 enzymes exhibit 100% amino acid sequence identity to the native h-PON1, with minimal changes necessary for increasing the catalytic activities. Using this approach, we were able to get a yield which is significantly higher than the yield of recombinant h-PON1 reported in the literature [24,28,50–53]. To the best of our knowledge, this is the first study that shows that h-PON1 (and its improved variant) can be produced in active form and can also give high yield by refolding the recombinant enzymes expressed as IBs in E coli. This approach can be used to develop an economical method for the industrial scale production of h-PON1 enzymes.

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Conflict of interest

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A patent application has been filed related to the products and technology described in this paper by the National Institute of Pharmaceutical Education and Research (NIPER), SAS Nagar. Abhay H Pande, Priyanka Bajaj, Rajan K Tripathy and Geetika Aggarwal are inventor in the patent application and hold an indirect interest in this intellectual property.

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Reference of submitted sequences

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The GenBank accession number of the submitted nucleotide sequences of rh-PON1(wt) and rh-PON1(H115W;R192K) are KC456197, KC456200, respectively.

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Acknowledgments

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This work was supported by the research grants to AHP from NIPER, SAS Nagar (NPLC-AHP). The authors are grateful to Prof.

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Please cite this article in press as: P. Bajaj et al., Expression and purification of biologically active recombinant human paraoxonase 1 from inclusion bodies of Escherichia coli, Protein Expr. Purif. (2015), http://dx.doi.org/10.1016/j.pep.2015.05.011

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Richard W. James (University Hospital, Geneva, Switzerland) for the gift of monoclonal mouse anti-HuPON1 antibody. Priyanka Bajaj (CSIR-SPM-SRF) and Geetika Aggarwal (CSIR-SRF) are thankful to CSIR, New Delhi for financial support in the form of CSIR Fellowship.

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References

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