Human recombinant prolidase from eukaryotic and prokaryotic sources Expression, purification, characterization and long-term stability studies Anna Lupi*, Sara Della Torre*, Elena Campari, Ruggero Tenni, Giuseppe Cetta, Antonio Rossi and Antonella Forlino Department of Biochemistry ‘Alessandro Castellani’, University of Pavia, Italy Extracellular and intracellular proteases perform essen- tial functions in all living organisms by both mediating nonspecific protein hydrolysis and acting as processing enzymes that perform highly selective, limited and effi- cient cleavage of specific substrates that influence many biological processes. In recent years, the availability of the human genome sequences, together with the devel- opment of powerful tools, such as genomics, proteo- mics and bioinformatics, has provided new possibilities for investigating the degradome, the complete set of proteases expressed at a specific moment or under cer- tain circumstances by a cell, tissue or organism. In a relatively recent review, Puerte et al. [1] listed 553 genes encoding proteases or homologous proteases in Keywords histidine tag; human recombinant prolidase; long-term enzyme stability; on-column tag removal; prolidase deficiency Correspondence A. Forlino, Department of Biochemistry ‘Alessandro Castellani’, Section of Medicine and Pharmacy, University of Pavia, Via Taramelli 3 ⁄ B, 27100 Pavia, Italy Fax: +39 0382 423108 Tel : +39 0382 987235 E-mail: aforlino@unipv.it *These authors contributed equally to this work (Received 27 June 2006, revised 10 October 2006, accepted 16 October 2006) doi:10.1111/j.1742-4658.2006.05538.x Prolidase is a Mn 2+ -dependent dipeptidase that cleaves imidodipeptides containing C-terminal proline or hydroxyproline. In humans, a lack of prolidase activity causes prolidase deficiency, a rare autosomal recessive disease, characterized by a wide range of clinical outcomes, including severe skin lesions, mental retardation, and infections of the respiratory tract. In this study, recombinant prolidase was produced as a fusion protein with an N-terminal histidine tag in eukaryotic and prokaryotic hosts and purified in a single step using immobilized metal affinity chromatography. The enzyme was characterized in terms of activity against different substrates, in the presence of various bivalent ions, in the presence of the strong inhib- itor Cbz-Pro, and at different temperatures and pHs. The recombinant enzyme with and without a tag showed properties mainly indistinguishable from those of the native prolidase from fibroblast lysate. The protein yield was higher from the prokaryotic source, and a detailed long-term stability study of this enzyme at 37 °C was therefore undertaken. For this analysis, an ‘on-column’ digestion of the N-terminal His tag by Factor Xa was per- formed. A positive effect of Mn 2+ and GSH in the incubation mixture and high stability of the untagged enzyme are reported. Poly(ethylene glycol) and glycerol had a stabilizing effect, the latter being the more effective. In addition, no significant degradation was detected after up to 6 days of incubation with cellular lysate. Generation of the prolidase in Escheri- chia coli, because of its high yield, stability, and similarity to native proli- dase, appears to be the best approach for future structural studies and enzyme replacement therapy. Abbreviations CHO, Chinese hamster ovary; IMAC, Ni ⁄ nitrilotriacetate-immobilized metal affinity chromatography; PD, prolidase deficiency; PEG, poly(ethylene glycol). 5466 FEBS Journal 273 (2006) 5466–5478 ª 2006 The Authors Journal compilation ª 2006 FEBS the human genome and catalogued 53 diseases caused by mutations in protease genes, mainly recessive loss of function mutations. Our understanding of the path- ophysiology of these diseases and the development of therapeutic approaches are based on a knowledge of the exact structure, function and regulation of the pathologically relevant proteases under physiological conditions. Prolidase (EC 3.4.13.9) is a member of the metallo- peptidase family, and the lack of prolidase activity, due to mutations in the prolidase gene, is responsible in humans for the recessive inherited disease prolidase deficiency (PD). Patients with PD are characterized by intractable skin ulceration, mainly on the lower limbs, various levels of mental retardation, and recurrent infections of the respiratory tract [2]. Sixteen mutations have been described as causes of PD [3–17], but the basis of the clinical outcome and the genotype ⁄ pheno- type relationship are still unclear and no definitive cure for the disease is available. Prolidase is widespread in nature and has been isola- ted from mammals [2], bacteria, such as Lactobacillus [18] and Xanthomonas [19], and from the archeon Pyrococcus furiosus [20]. Human prolidase is a man- ganese-dependent exopeptidase. It is a unique enzyme being the only one able to hydrolyze the peptide bond in iminodipeptides containing C-terminal proline or hydroxyproline, because of the specific conformation that the peptide chain assumes in the presence of the pyrrolidine side chain of proline residues [21]. The structure of prolidase and its catalytic site, as well as the catalytic mechanism of the mammalian enzyme, have not yet been defined. Prolidase is involved in the final stage of the cata- bolism of proline-rich and hydroxyproline-rich dietary and endogenous proteins, such as collagen. It supplies and recycles proline for protein synthesis and cell growth [2]. It has also been reported that prolidase guarantees proline availability as a substrate for gener- ating reactive oxygen species by proline oxidase during proline-induced apoptosis [22–24]. Phang and cowork- ers recently demonstrated that NO stimulates prolidase activity and suggested an interaction between inflam- matory signalling pathways and regulation of the ter- minal step of matrix degradation [25]. Furthermore, prolidase has a biotechnological rele- vance not related to its physiological function. It can be used as a cheese-ripening agent, as proline released from proline-containing peptides in cheeses reduces their bitterness, making this enzyme particularly appealing to the dietary industry [26]. It has also been reported that prolidase is similar to organophos- phorus acid anhydrolase which hydrolyses highly toxic organophosphorus acetylcholinesterase inhibitors, including various chemical warfare agents and pesti- cides. This function makes the enzyme relevant in detoxification strategies [27,28]. Here we describe the synthesis, purification and bio- chemical characterization of human recombinant proli- dase from eukaryotic and prokaryotic hosts and compare the enzyme with the endogenous prolidase of human fibroblasts. The similarity of the recombinant and endogenous prolidase in terms of substrate specif- icity, optimal pH and temperature for activity, and metal dependence is discussed, and a detailed analysis of long-term enzyme stability at 37 °C is presented. These properties are particularly important for struc- tural studies and developing strategies for enzyme replacement therapy for PD. Results Recombinant human prolidase expression and purification from eukaryotic and prokaryotic sources Total cellular RNA from cultured normal human fibroblasts was reverse-transcribed, and prolidase cDNA was amplified by PCR with specific primers. The amplified product was subcloned into the eukary- otic expression vector pcDNA4 ⁄ HisMax (pcDNA4 ⁄ HisMax-prol) and into the prokaryotic expression vec- tor pET16b (pET16b-prol) (Fig. 1A,B). Chinese hamster ovary (CHO) cells were transfected using Lipofectamine. Stable transfected cells were obtained after selection with Zeocin for 8 days. Escherichia coli cells were transformed by heat shock, following a standard protocol. Ampicillin was used as antibiotic for selection. CHO culture conditions were optimized to obtain the highest yield of recombinant enzyme. For this, 2 · 10 6 stable transfected CHO cells were plated in T175 flasks and harvested after 24, 48, 96 and 120 h at 37 °C and 5% CO 2 . We observed a progressive increase in the total protein content over time (0.19 mgÆmL )1 at 24 h, 0.45 mgÆmL )1 at 48 h, 4.8 mgÆmL )1 at 96 h and 7.45 mgÆmL )1 at 120 h), but the highest prolidase activity was observed after 96 h (3.42 lmol Gly-Pro idrolÆh )1 ÆmL )1 ); all purifications were therefore performed after 96 h of growth. As the recombinant protein contained an N-terminal His tag with 6 histidine residues, we used Ni ⁄ nitrilotriacetate- immobilized metal affinity chromatography (IMAC) with an imidazole step gradient from 50 to 500 mm to purify the enzyme. Recombinant prolidase was elu- ted at an imidazole concentration of 200–300 mm, A. Lupi et al. Human recombinant prolidase from CHO and E. coli FEBS Journal 273 (2006) 5466–5478 ª 2006 The Authors Journal compilation ª 2006 FEBS 5467 accounting for 14% of total prolidase activity. The fractions containing the recombinant enzyme were dia- lysed for 24 h against 50 mm Tris ⁄ HCl (pH 7.8) ⁄ 4mm 2-mercaptoethanol at 4 °C to eliminate imidazole and NaCl, which acted as prolidase inhibitors at the con- centrations used (data not shown). The recombinant protein was identified by western blotting using an HisG antibody (Fig. 2A,B). The recombinant enzyme had a molecular mass of 58 kDa. Escherichia coli BL21 (DE3) bacteria transformed with the prokaryotic vector pET16b-prol, allowed us to obtain large amounts of the recombinant enzyme; from 1 L bacterial culture we purified about 8 mg recombinant prolidase able to hydrolyze 18 g Gly-Pro dipeptide in 1 h. Upon cell growth, bacteria were pell- etted, lysed as described in Experimental procedures, and loaded on to an IMAC column for purification as the recombinant enzyme expressed in bacteria also had an N-terminal His tag. Recombinant prolidase was recovered at high imidazole concentrations (250 and 300 mm), because of the presence of 10 His residues in the tag. We recovered about 20% of the total lysate prolidase activity. Fractions containing the recombin- ant enzyme were pooled and dialysed for 24 h against 50 mm Tris ⁄ HCl, pH 7.8, containing 4 mm 2-merca- ptoethanol at 4 °C to eliminate imidazole and NaCl. The elution fractions were analyzed by SDS ⁄ PAGE with Coomassie Blue staining, and the purified protein was identified by western blotting using an Penta-His antibody (Fig. 2C–E). The recombinant enzyme had a molecular mass of 57 kDa. His-tag cleavage of recombinant human prolidase To evaluate the influence of the His-tag on enzyme activity and stability, we characterized the recombinant A BDE C Fig. 2. (A, C) Recombinant human prolidase purification. Elution profile of recombinant prolidase obtained from CHO (A) and E. coli (C). j, prolidase activity; r, gradient of imidazole. (B, D) Western blotting of recombinant prolidase obtained from CHO (B) and E. coli (D) using antibody against the histidine tag. (E) Coomassie blue-stained SDS ⁄ polyacrylamide gel of purified recombinant prolidase from E. coli. Fig. 1. Expression vectors for recombinant prolidase. (A) Eukaryotic expression vector pcDNA4 ⁄ HisMax containing an N-terminal His tag and the sequence for the enterokinase cleavage site (EK site) in-frame with the human prolidase sequence under the control of the cytomegalo- virus (CMV) promoter and the SP163 enhancer. (B) Prokaryotic expression vector pET16b containing an N-terminal His tag and the sequence for the Factor Xa cleavage site in-frame with the human prolidase sequence under the control of the T7 promoter. Human recombinant prolidase from CHO and E. coli A. Lupi et al. 5468 FEBS Journal 273 (2006) 5466–5478 ª 2006 The Authors Journal compilation ª 2006 FEBS enzyme in both the presence and absence of the poly- histidine tag. The tag was removed from the recombin- ant enzymes obtained from CHO cells and E. coli cells by enterokinase and by Factor Xa digestion, respect- ively, using in-batch digestion, as described in Experi- mental procedures. Western blotting analysis using a specific antibody against the His tag was performed to evaluate the cleavage (Fig. 3A,B). To produce higher amounts of untagged recombin- ant protein for the stability study, we optimized an ‘on-column’ digest of the recombinant prolidase obtained from E. coli. About 6 mg tagged protein was loaded on a column containing 1 mL Ni ⁄ nitrilotriace- tate resin. Then 24 U Factor Xa was added, and the column was incubated at 35 °C for 72 h. The elution was performed with an imidazole step gradient (20– 500 mm). The cleaved protein was eluted at 20–50 mm imidazole as shown by SDS ⁄ PAGE and western blot- ting using a specific Penta-His antibody (Fig. 3C,D). The total cleavage efficiency was > 80%, and the first three fractions contained 100% of the digested proli- dase detectable by SDS ⁄ PAGE, but not western blot- ting (Fig. 3C,D). The < 20% uncleaved enzyme was digested more than 50% with a second digestion performed using the same conditions. N-Terminal sequence The sequence of the N-terminal 25 amino acids of the recombinant prolidase purified from E. coli was unequivocally determined by automated Edman degra- dation. We analyzed the enzyme both with and with- out histidine tag. Comparison of the first 25 amino acids of the tagged purified protein (GHHHHHHHH HHSSGHIEGRHMAAAT) with that of the His- tagged human recombinant prolidase, predicted from the nucleotide sequence, revealed an exact match for 92% of the total protein analysed. Furthermore, the N-terminal sequence of the first 25 amino acids of the His tag-cleaved protein (HMAAATGPSFWLGNE TLKVPLALFA) showed an exact match with the expected residues. The same analysis was attempted for the prolidase purified from CHO cells, but no clear results were obtained because of the limited amount of purified protein. Substrate specificity, inhibitory effect of Cbz-Pro, optimum temperature, pH and metal dependence We compared functional properties of the recombinant prolidases (purified from CHO cells or E. coli both with a His tag and after His tag cleavage) with endo- genous human prolidase from fibroblast lysate. Substrate specificity studies confirmed Gly-Pro as the preferred substrate for prolidase. Both recombinant enzymes and wild-type prolidase hydrolysed the proline dipeptides tested with at least the same efficiency (Gly- Pro > Ala-Pro > Phe-Pro > Leu-Pro) (Table 1). Cbz-Pro is a well known in vitro and in vivo human prolidase inhibitor [29]. Activity studies confirmed A B C D Fig. 3. Western blotting analysis of the removal of the polyhistidine tag from recombinant prolidase obtained from CHO (A) and from E. coli (B). +His, control samples; –His, cleaved samples. SDS ⁄ PAGE (C) and western blotting (D) of the fractions eluted after digestion ‘on-column’ by Factor Xa of the N-terminal His tag of the recombinant prolidase obtained from E. coli. FT, Flow through, con- taining 20 m M imidazole; 50, 100 and 250 mM imidazole concentra- tions were used for the elution. Table 1. Recombinant and wild-type prolidase activity tested against different substrates. The percentage was calculated taking as 100% prolidase activity against Gly-Pro. Data represent the mean ± SD from three independent determinations. CHOprol + His, CHOprol ) His, recombinant prolidase from CHO cells with and without the His tag; E. coliprol + His, E. coliprol ) His , recombinant prolidase from E. coli with and without the His tag; FBprol, endogenous fibroblast prolidase. Prolidase activity (%) CHOprol + His CHOprol – His E. coliprol + His E. coliprol – His FBprol Ala-Pro 56.25 ± 1.820 71.89 ± 4.925 31.71 ± 2.276 33.16 ± 1.798 44.34 ± 1.438 Phe-Pro 23.39 ± 3.339 20.74 ± 1.254 17.53 ± 3.783 27.66 ± 5.492 20.20 ± 4.452 Leu-Pro 5.12 ± 1.262 4.68 ± 0.190 0.88 ± 0.433 0.28 ± 0.158 5.69 ± 0.564 A. Lupi et al. Human recombinant prolidase from CHO and E. coli FEBS Journal 273 (2006) 5466–5478 ª 2006 The Authors Journal compilation ª 2006 FEBS 5469 Cbz-Pro as an inhibitor also of the recombinant enzymes. No significant differences were revealed between recombinant tagged and untagged and wild- type prolidase with a 1 : 4 Cbz-Pro⁄ Gly-Pro molar ratio (Table 2). A temperature ⁄ activity profile was constructed by measuring enzyme activity towards Gly-Pro substrate over a range of 60 °C (at 20, 37, 50, 70, 80 °C), with the optimum activity at 50 °C (the activity at this tem- perature was assumed to be 100%). At 37 °C, the activity of the recombinant His-tagged enzyme was 50% of the maximum activity measured at 50 °C, and the activity of the same enzyme without the tag was 60–70%, closer to the 80% activity of the endogenous enzyme (Fig. 4A). The maximum activity of the recombinant and wild- type enzyme, determined by measuring enzyme activity towards Gly-Pro as substrate, was at pH 7.8 (the activ- ity at this pH was assumed to be 100%) (Fig. 4B). The effect of various bivalent ions (Ca 2+ ,Co 2+ , Mg 2+ ,Mn 2+ and Zn 2+ ) on prolidase activity was tes- ted only for the recombinant enzyme produced in E. coli because of difficulties in obtaining sufficient amounts of pure protein from the eukaryotic source without MnCl 2 preactivation. The maximum activity was obtained with Mn 2+ (assumed to be 100%). Less than 30% of the activity was obtained with the other metals tested (Fig. 4C). Long-term stability at 37 °C Owing to the higher amount of the recombinant proli- dase obtained from E. coli, the simple purification method described, and its potential use for replace- ment therapy, we focused on the long-term thermosta- bility at 37 °C of this enzyme. We tested both tagged and untagged enzymes to study whether the His tag affected the activity over time. The recombinant proli- dase was preactivated for 1 h at 50 °C in presence of MnCl 2 and GSH and incubated at 37 °C. The activity was evaluated daily using Gly-Pro as substrate; the activity on day 0 was considered to be 100%. On day 1, the untagged prolidase had a higher activ- ity (70%) than the tagged enzyme (47%) (P<0001), but both enzymes had lost almost total activity by day 2. No significant difference was detected at this time between the tagged and untagged prolidase (7% and 13%, respectively, P ¼ 0.103) (Fig. 5A). To improve enzyme activity over time, we incubated the preactivated recombinant enzymes at 37 °C in pres- ence of GSH or of GSH and MnCl 2 . The presence of GSH partially recovered the loss of enzyme activity previously detected. In fact, on day 2, the tag- ged enzyme had 55% of the initial activity and the untagged prolidase 66% (Fig. 5B). The addition of MnCl 2 and GSH showed the most promising effects in stabilizing and activating the enzyme. Both tagged and untagged enzymes showed an increase in activity on day 1 (160% and 154%, respectively, P ¼ 0.395). On day 2, the activity of the tagged enzyme was 87%, whereas the untagged recom- binant prolidase had conserved 159% of the activity (P<0.001). By day 3, the activity of the tagged enzyme was only 27%, whereas the activity of the untagged enzyme was significantly higher (127%; P<0.001) (Fig. 5B). In an attempt to stabilize further the recombinant prolidase obtained from E. coli, we tested the effects of two molecules often used as stabilizing agents [poly(ethylene glycol) (PEG)200 and glycerol], gener- ating, respectively, hydrophobic and hydrophilic inter- action with the protein [30,31]. We focused on the untagged enzyme because the experiments described above showed that it was more active for a long time and because of the potential, although not demonstra- ted, for the immunological reaction caused by the tag to be used in future therapeutic applications. While PEG reproduced the effect of Mn 2+ and GSH incuba- tion, the glycerol had a strong activating effect up to day 6 (561% of the initial activity) (Fig. 5C). We next tested in vitro the long-term stability at 37 °C of the untagged enzyme in the presence of a cel- lular lysate without proteinase inhibitors, collected from a prolidase-deficient patient and lacking endo- genous prolidase activity. No significant difference was detected at any time (from day 1 to day 6) between the activity of the recombinant prolidase in the presence or absence of the intracellular lysate (Fig. 6). Table 2. Inhibitory effect of Cbz-Pro on recombinant and wild-type prolidase activity. A 1 : 4 Cbz-Pro ⁄ Gly-Pro molar ratio was used. The per- centage was calculated taking as 100% prolidase activity against Gly-Pro. Data represent the mean ± SD from three independent determina- tions. Prolidase activity (%) CHOprol + His CHOprol – His E. coliprol + His E. coliprol – His FBprol Cbz-Pro ⁄ Gly-Pro 34.48 ± 9.966 36.34 ± 7.992 36.85 ± 2.474 33.81 ± 2.259 42.39 ± 7.998 Human recombinant prolidase from CHO and E. coli A. Lupi et al. 5470 FEBS Journal 273 (2006) 5466–5478 ª 2006 The Authors Journal compilation ª 2006 FEBS Finally, we tested the long-term thermostability of the recombinant prolidase obtained from CHO cells and the endogenous fibroblast prolidase, using the con- ditions found to be optimal for the enzyme produced in E. coli (37 °C in the presence of MnCl 2 and GSH). After 8 days, the activity of the endogenous enzyme and the tagged and untagged recombinant prolidase was close to the activity measured on day 1 (123%, 90% and 85%, respectively). Prolidase localization Cellular localization was evaluated in stable transfect- ed CHO cells. Cytoplasmic and nuclear fractions were separated by centrifugation, and both fractions were treated to purify recombinant prolidase by affinity chromatography as previously described. The fractions eluted at 300 mm, which should contain the recombinant enzyme, were pooled and dialysed. Activity was mainly detected in the fractions obtained from the cytosolic samples. We also analyzed the eluted cytosol and nuclei samples by western blotting with the HisG antibody. The recom- binant prolidase was present mainly in the cytosol (Fig. 7). Discussion This paper describes the purification and characteriza- tion of a dipeptidase, human recombinant prolidase, expressed in eukaryotic and prokaryotic hosts. We compared the substrate specificity, optimum tempera- ture and pH, the inhibitory effect of Cbz-Pro, the metal dependence, and the long-term activity at 37 °C of the recombinant enzymes and the endogenous proli- dase present in cellular fibroblast lysates. The recombinant enzymes from both sources were obtained as fusion proteins with an N-terminal poly His tag, which allowed a one-step purification proce- dure by affinity chromatography, but which could compromise the enzyme properties and ⁄ or its potential therapeutic use, as pointed out by Jenny et al. [32]. All the characterization experiments were performed on enzymes with the histidine tag (tagged) and after speci- fic tag cleavage (untagged). Two different methods were used for tag removal. For small-scale production, a digestion was per- formed in-batch with enterokinase for the recombin- ant prolidase from eukaryotic cells, and with Factor Xa for the protein synthesized in E. coli. For large- scale production of untagged recombinant enzyme, required for the stability studies and for future struc- tural and pharmacological applications, we optimized an ‘on-column’ digestion of the prolidase produced in E. coli using Factor Xa. It is the first report of this method applied to an N-terminal tagged protein using this enzyme. Cleavage of the affinity tags while the target protein was still bound to the affinity column allowed us to obtain over 80% of the com- pletely (100%) cleaved protein in a single chromato- graphic step. By SDS ⁄ PAGE, the purified enzymes from CHO cells and E. coli were shown to have a molecular mass Fig. 4. Effect of temperature (A), pH (B) and metals (C) on the activity of recombinant prolidase from CHO cells with or without the His tag (CHOprol + His, CHOprol ) His), recombinant prolidase from E. coli with and without the His tag (E. coliprol + His, E. colip- rol ) His) and endogenous fibroblast prolidase (FBprol). Data are expressed as mean ± SD from three experiments. A. Lupi et al. Human recombinant prolidase from CHO and E. coli FEBS Journal 273 (2006) 5466–5478 ª 2006 The Authors Journal compilation ª 2006 FEBS 5471 of 58 kDa and 57 kDa, respectively, both very close to the values of 58.094 kDa and 57.121 kDa estimated by the SwissProt DataBase based on the enzyme sequences, and to the molecular mass reported for human prolidase from different sources [33–35]. The amount of recombinant enzyme obtained from CHO cells was not quantifiable at the protein level, and its activity, expressed as mmol Gly-Pro hydro- lyzed in 1 h on total proteins present in the cellular lysate, was 1000-fold lower than that of recombinant prolidase obtained from E. coli (0.85 · 10 )3 ± 1.2 · 10 )3 mmolÆh )1 Æmg )1 and 1.315 ± 0.525 mmolÆ h )1 Æmg )1 , respectively). This difference may be due to the different promo- ter used, the different copy number of the exogenous DNA, or the fact that eukaryotic cells could tolerate only a limited amount of prolidase in their cyto- plasm. From literature data, mammalian prolidase is min- imally (0.5%) glycosylated and up-regulated by phos- phorylation at serine ⁄ threonine ⁄ tyrosine residues [25]. Although because of the low amount of prolidase obtained from the eukaryotic host, we were unable to Fig. 5. Activity of recombinant prolidase pro- duced in E. coli after long-term incubation at 37 °C. (A) Percentage of activity of tagged (+ His) and untagged (– His) enzyme; (B) percentage of activity of tagged and untagged enzyme incubated in the presence of GSH (+ His ⁄ GSH, – His ⁄ GSH) or GSH and Mn 2+ ions (+ His ⁄ GSH, Mn 2+ , – His ⁄ GSH, Mn 2+ ); (C) percentage of activity of the untagged enzyme in the presence of glycerol and PEG200 (– His ⁄ GSH, Mn 2+ , 25% glycerol; – His ⁄ GSH,Mn 2+ , 25% PEG). The activity on day 0 is assumed to be 100%. Data are expressed as mean ± SD from three experiments. d, Day of incubation. Fig. 6. Long-term activity of the untagged recombinant prolidase obtained from E. coli in the absence and presence of PD fibroblast cellular lysate. Data are expressed as mean ± SD from three experiments. d, Day of incubation. Fig. 7. Western blotting analysis using HisG antibody to evaluate the cellular localization of recombinant prolidase produced in trans- fected CHO cells. C, Cytosol; N, nucleus. Human recombinant prolidase from CHO and E. coli A. Lupi et al. 5472 FEBS Journal 273 (2006) 5466–5478 ª 2006 The Authors Journal compilation ª 2006 FEBS test directly the effect of post-translational modifica- tions on enzyme activity, our data on the recombinant enzyme suggest that the lack of glycosylation and phosphorylation does not greatly reduce prolidase activity, making possible its production in a prokary- otic host in high amounts and at low cost. The substrate specificity of both tagged and untagged enzymes obtained from eukaryotic and prok- aryotic sources was indistinguishable from that of endogenous fibroblast prolidase, the preferred sub- strate being Gly-Pro, followed by Ala-Pro. This is in accordance with data previously reported for human endogenous prolidase from fibroblasts and erythro- cytes [33–40]. The catalytic activity of both recombinant and endogenous prolidase showed virtually identical responses to changes in temperature and pH. All enzymes showed a temperature optimum for activity of 50 °C and a pH optimum of 7.8. However, at the phy- siological temperature of 37 °C, higher activity was detected for the endogenous enzyme, and the tagged recombinant prolidase showed the lowest activity, although the differences were not significant. Human prolidase isolated from different tissues requires Mn 2+ ions [33], and this metal cannot be effectively replaced by other metals. Similarly, our recombinant enzyme showed highest activity in the presence of MnCl 2 , with activities of less than 30% in the presence of Ca 2+ ,Co 2+ ,Mg 2+ and Zn 2+ . Fur- thermore, preactivation with Mn 2+ before purification by IMAC was a requirement for the enzyme synthes- ized in CHO cells; no binding to the Ni 2+ was possible without preactivation (data not shown). The amount of purified recombinant prolidase from E. coli was increased by including MnCl 2 in the culture medium and preactivating the enzyme with Mn 2+ before IMAC purification. These observations suggest that the metal is not only necessary for enzyme activity but also involved in its folding. The native and recombinant form of human fibro- blast prolidase exhibited essentially equivalent physical and catalytic properties, but, considering the high yield, properties, and low cost of the enzyme from E. coli, the recombinant prolidase obtained from this prokaryotic source appeared more attractive for struc- tural studies and therapeutic purposes, so a detailed investigation of its stability was undertaken. We dem- onstrated that the addition of GSH and Mn 2+ ions had both a stabilizing and activating effect, which per- sisted up to 3 days for the untagged enzyme. Further investigation showed that the enzyme without a His tag was stabilized and stimulated by PEG, a stabilizer often used for protein replacement therapy, but better results were obtained using glycerol. The presence of 3.2 m glycerol allowed an activity of 561% with respect the initial tested activity after 6 days of incuba- tion at 37 °C. As enzyme replacement therapy implies that the therapeutic molecules will be in contact with the intra- cellular environment, we also tested the stability of our recombinant enzyme ex vivo and demonstrated that it is not digested by endogenous proteinase present in fibroblast lysate up to 6 days. Few attempts have been made so far to develop an enzyme replacement therapy for PD. Genta et al. [41] encapsulated porcine kidney prolidase stabilized by MnCl 2 , GSH and BSA in a biodegradable micro- sphere. This system allowed greater release of the enzyme in vitro after 20 h of incubation at 37 °C, but basically no activity was detected after 40 h. In an ex vivo experiment in the presence of fibroblast cell lysate, the activity was detectable only up to 4 days. Interestingly, pig prolidase was not stable at all without micro-encapsulation, losing its activity both in vitro and ex vivo in 4 h [41]. The same approach was used to deliver the enzyme in human cultured fibroblasts from controls and patients with PD [42]: a 49% increase in prolidase activity after 3 days of incubation was detected. Recently, porcine kidney prolidase was encapsulated in liposomes to deliver the enzyme to cultured fibroblasts: the maxi- mum release of enzyme activity was on day 6 of incubation [43]. On the basis of these data, the availability of a new recombinant prolidase with the same human sequence, easily produced in high amounts, without need of exo- genous BSA as a stabilizer, offers a new valuable tool for testing both the delivery systems already under study and for developing new ones. Furthermore, the recombinant enzyme will allow detailed structural studies, which should lead to a bet- ter understanding of prolidase function and regulation. Experimental procedures Cell strains and culture conditions Primary dermal control fibroblasts and CHO cells were purchased from International Pbi SpA (Milan, Italy) and American Type Culture Collection (ATCC), respectively. Cells were grown at 37 °C in the presence of 5% CO 2 in Dulbecco’s modified Eagle’s medium or RPMI 1640, respectively (Sigma, St Louis, MO, USA), supplemented with 10% fetal calf serum (Euroclone, Pero, Italy). Fibroblasts were used between the fourth and tenth passage. A. Lupi et al. Human recombinant prolidase from CHO and E. coli FEBS Journal 273 (2006) 5466–5478 ª 2006 The Authors Journal compilation ª 2006 FEBS 5473 Escherichia coli BL21 (DE3) [F – ompT hsdS B (r B – m B – )gal dcm (DE3)] bacteria were grown in Luria-Bertani medium in an orbital incubator at 37 °C agitated at 190 r.p.m. Construction of eukaryotic expression vector and CHO cell transfection Total cellular RNA, isolated from cultured human control fi- broblasts, was extracted by TriReagent (Molecular Research, Cincinnati, OH, USA); 1 lg was reverse-transcribed using the Gene Amp Gold RNA PCR kit according to the manu- facturer’s protocol. The forward primer 5¢-AGGTACCT ATGGCGGCGGCCACCGGACCCT-3¢ (nucleotides 17– 38) and the reverse primer 5¢-GATCTAGATGATTCT GGGTGCCGTCTCTCGCTAC-3¢ (nucleotides 1582–1607), carrying 5¢ overhang sequences containing the restriction sites for KpnI and XbaI, respectively, were used to amplify by PCR the human prolidase cDNA (GenBank NM_000285). PCR amplification was carried out at 94 °C for 2 min, 35 cycles at 94 °C for 30 s, 65 °C for 30 s, and 68 °C for 2 min, and a final extension step at 72 °C for 10 min. The amplified product was gel-purified using Nucleo Spin Extract (Macherey-Nagel, Du ¨ ren, Germany), digested at 37 °C with 10 U KpnI and XbaI (Invitrogen), ligated into KpnI ⁄ XbaI-digested pcDNA4 ⁄ HisMax eukaryotic expres- sion vector (Invitrogen, Carlsbad, CA, USA) using T4-DNA ligase, and transformed into E. coli TOP10 competent cells. This vector contains the strong cytomegalovirus (CMV) pro- moter and a SP193 enhancer, the sequence coding for an N-terminal polyhistidine tag, and the sequence for the enter- okinase-recognition site 5¢- to the polylinker site. The vector also contains the genes for resistance to Zeocin. The inserted sequence was confirmed by sequencing (see below for details). CHO cells were plated at 3 · 10 5 cell density in 60-mm Petri dishes and transfected with 8 lg pcDNA4 ⁄ HisMax- prol using Lipofectamine 2000 Reagent (Invitrogen) follow- ing the manufacturer’s suggestion. Transfected cells were selected with 0.3 mgÆmL )1 Zeocin for 8 days to obtain a stable transfected cell line. Construction of prokaryotic expression vector and E. coli BL21 (DE3) transformation Total cellular RNA from cultured human control fibroblasts (1 lg) was reverse-transcribed using the Gene Amp Gold RNA PCR kit as previously described. The forward primer 5¢-AGGCATATGGCGGCGGCCACCGGACCCT-3¢ (nu- cleotides 17–38) and the reverse primer 5¢-GACGGATC CATTCTGGGTGCCGTCTCTCGCTAC-3¢ (nucleotides 1582–1607), carrying 5¢ overhang sequences containing the restriction sites for NdeI and BamHI, respectively, were used to amplify the prolidase cDNA (GenBank NM_000285) by PCR. PCR amplification was carried out under the condi- tions described above. The amplified product was gel-puri- fied using Nucleo Spin Extract, digested with 10 U NdeI and BamHI, ligated into the prokaryotic-expressing plasmid pET16b (Novagen, San Deigo, CA, USA), previously diges- ted with the same restriction enzymes. This vector contains the strong T7 promoter, the sequence coding for an N-ter- minal polyhistidine tag followed by the sequence for the Factor Xa-recognition site 5¢- to the polylinker site. The vec- tor also contains the gene for ampicillin resistance. The inserted sequence was confirmed by sequencing. Escherichia coli BL21 (DE3) cells were then transformed by heat shock for protein expression and purification. DNA sequencing DNA sequencing was performed with the ABI-Prism Big- Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin–Elmer, Monza, Italy). An automatic sequencer ABI-Prism 310 (Perkin–Elmer) was used. Purification of CHO recombinant human prolidase Stable transfected CHO cells were plated at 2 · 10 6 cell density in T175 flasks and grown for 96 h in RPMI 1640 supplemented with 10% fetal bovine serum. Upon medium removal and three NaCl ⁄ P i washes, the cell layer was mechanically removed. Pelleted cells were suspended in 50 mm Tris ⁄ HCl (pH 7.8) ⁄ 4mm 2-mercaptoethanol, soni- cated on ice, and centrifuged at 16 000 g for 30 min at 4 °C. The lysate was incubated with 0.75 mm GSH and 1mm MnCl 2 for 12 h at 4 °C, then dialysed overnight at 4 °C against 50 mm Tris ⁄ HCl (pH 7.8) ⁄ 4mm 2-mercapto- ethanol. Imidazole and NaCl were added to a final concen- tration of 0.01 m and 0.3 m, respectively. An aliquot of 1 mL of 50% Ni ⁄ nitrilotriacetate agarose (Qiagen, Milan, Italy) slurry was added to the lysate and mixed gently by shaking at 4 °C for 60 min. Then the col- umn flow-through was collected, and a wash step was per- formed using 50 mm Tris ⁄ HCl, 300 mm NaCl, 4 mm 2-mercaptoethanol (elution buffer) and 20 mm imidazole. For elution of recombinant protein, the following step gra- dient from 50 mm to 500 mm imidazole in elution buffer was used: 3 mL elution buffer with 50 mm imidazole, 1 mL elution buffer with 100 mm imidazole, three aliquots of 0.33 mL of the same buffer with 200 mm imidazole, three aliquots of 0.33 mL of elution buffer with 300 mm imidaz- ole, and finally two aliquots of 0.5 mL of elution buffer containing 500 mm imidazole. Prolidase activity was deter- mined for each fraction collected. Enzyme-containing frac- tions were pooled and dialysed for 24 h against 50 mm Tris ⁄ HCl (pH 7.8) ⁄ 4mm 2-mercaptoethanol at 4 °C. The purified enzyme was stored at )80 °C in 0.3-mL aliquots in LoBind tubes (Eppendorf, Milan, Italy). Human recombinant prolidase from CHO and E. coli A. Lupi et al. 5474 FEBS Journal 273 (2006) 5466–5478 ª 2006 The Authors Journal compilation ª 2006 FEBS Purification of E. coli BL21 (DE3) recombinant human prolidase Escherichia coli BL21 (DE3)-transformed bacteria were grown in Luria-Bertani medium containing 50 lgÆmL )1 ampicillin and 1 mm MnCl 2 at 37 °C in an orbital incuba- tor and agitated at 190 r.p.m. At A 600 ¼ 0.6, the cells were induced with 1 mm isopropyl b-d-1-thiogalactopyranoside for 2 h at 37 °C. Bacteria were pelleted by centrifugation at 5000 g for 15 min (RC5C Plus centrifuge, SS34 rotor, Sorvall, New- town, CT, USA) and washed twice in 10 mm Tris ⁄ HCl (pH 7.8) ⁄ 150 mm NaCl ⁄ 1mm EDTA ⁄ 4mm 2-mercaptoeth- anol ⁄ 4mm benzamidine and resuspended in 2 mL of the same buffer. Cells were treated with lysozyme (100 mgÆmL )1 ) on ice for 1 h, then lysed by addition of N-laurylsarcosine to 1.3% final concentration. After being vortex-mixed for 5 s, cells were sonicated, then Triton X-100 was added to 2% final concentration. The lysate was clarified by centrifugation for 60 min at 23 500 g at 4 °C. Supernatants were filtered on 0.22-lm filters and diluted fivefold with 20 mm Tris ⁄ HCl (pH 7.8) ⁄ 4mm 2-mercaptoethanol. The solution containing the recombinant prolidase was preactivated by incubation with GSH (0.75 mm) and MnCl 2 (1 mm) for 1 h at 50 °C. The Mn 2+ excess was eliminated by dialysing the sample for 24 h at 4 °C against 20 mm Tris ⁄ HCl (pH 7.8) ⁄ 4mm 2-mercaptoethanol. Imidazole and NaCl were added to a final concentration of 0.01 m and 0.3 m, respectively. Ni ⁄ nitrilotriacetate agarose was used to purify the recom- binant protein using an imidazole step gradient as previ- ously described. Protein content and prolidase activity were evaluated for each fraction collected. Enzyme-containing fractions were pooled and dialysed for 24 h against 50 mm Tris ⁄ HCl (pH 7.8) ⁄ 4mm 2-mercaptoethanol at 4 °C. The purified enzyme was stored at )80 °C in 0.3-mL aliquots in LoBind tubes (Eppendorf). Endogenous prolidase Control fibroblasts were plated at a cell density of 8 · 10 5 in T75 tissue culture flasks and grown for 8 days in DMEM. After medium removal, the cell layer was washed with NaCl ⁄ P i , mechanically removed in 50 mm Tris ⁄ HCl (pH 7.8) ⁄ 4mm 2-mercaptoethanol, and sonicated. The lysate was then centrifuged at maximum speed, and the supernatant used as source of endogenous prolidase. His-tag cleavage His-tag digestion of purified recombinant prolidase from CHO cells was performed with recombinant enterokinase (Invitrogen) at 20 °C for 16 h; the recombinant entero- kinase was removed with EKapture agarose (Invitrogen) following the manufacturer’s suggestions. Digested and undigested prolidase were analyzed by western blotting (see below for details). His-tag digestion of purified recombinant prolidase obtained from E. coli was performed with Factor Xa (Nov- agen) at 35 °C for 16 h, and the Factor Xa was removed with Xarrest agarose (Novagen) following the manufac- turer’s suggestions. Digested and undigested prolidase were analyzed by western blotting (see below for details). For long-term stability studies, on-column digestion was performed modifying the method of Abdullah & Chase [44]. Briefly Ni ⁄ nitrilotriacetate agarose was equilibrated with 20 mm Tris ⁄ HCl (pH 7.8) ⁄ 300 mm NaCl (IMAC buf- fer) ⁄ 20 mm imidazole. E. coli recombinant protein ( 6 mg) in 50 mm Tris ⁄ HCl (pH 7.8) ⁄ 4mm 2-mercaptoethanol was added with NaCl, CaCl 2 and imidazole to a final concentra- tion of 100 mm,5mm and 20 mm, respectively, and loaded on the column in the presence of 4 UÆmg )1 Factor Xa. The column was incubated at 35 °C with shaking for 72 h. At the end of the incubation, the flow-through was collected followed by a step gradient of imidazole (50 mm, 100 mm , 250 mm and 500 mm) in IMAC buffer. The flow-through and fractions containing the untagged prolidase were identi- fied by SDS ⁄ PAGE and western blotting. The Factor Xa was removed with Xarrest agarose. Prolidase assays Prolidase activity was determined by the procedure of Myara et al. [45]. Commercially available proline was used for the standard curve. Briefly, prolidase activity in cell extract was assayed in 50 mm Tris ⁄ HCl, pH 7.8, after incu- bation with 1 mm MnCl 2 , 0.75 mm GSH and 100 mm gly- cyl-l-proline (Gly-Pro; MP Biomedicals, Milan, Italy) at 37 °C for 1 h. To evaluate the substrate specificity, a variety of sub- strates (ICN Biomedicals, Illkirch, France) were used: 100 mm glycyl-l-proline, 100 mm alanyl-l-proline, 100 mm leucyl-l-proline, 25 mm phenylalanyl-l -proline. To evaluate the inhibitory effect on prolidase, N-benzyl- oxycarbonyl-l-proline (Cbz-Pro; ICN Biomedicals) was used at a final concentration of 25 mm in the presence of 100 mm Gly-Pro. The following buffers substituted for standard assay buf- fer for determination of the enzyme’s pH ⁄ activity profile: 50 mm sodium acetate (pH 4.0), 50 mm sodium phosphate (pH 5.6), 50 mm Tris (pH 9.0). The temperature of optimum activity was evaluated by performing the assay at different temperatures (20, 37, 50, 70, 80 °C). Metal ion dependence was determined by incubating with different bivalent ions [Ca 2+ ,Co 2+ ,Mg 2+ ,Mn 2+ and Zn 2+ (salt stock solution 100 mm)] the enzyme obtained from E. coli without preactivation with Mn 2+ and fibroblast lysate. A. Lupi et al. Human recombinant prolidase from CHO and E. coli FEBS Journal 273 (2006) 5466–5478 ª 2006 The Authors Journal compilation ª 2006 FEBS 5475 [...]... compilation ª 2006 FEBS 5477 Human recombinant prolidase from CHO and E coli A Lupi et al 28 Wang SH, Zhi QW & Sun MJ (2005) Purification and characterization of recombinant human liver prolidase expressed in Saccharomyces cerevisiae Arch Toxicol 79, 253–259 29 Lupi A, Rossi A, Vaghi P, Gallanti A, Cetta G & Forlino A (2005) N-Benzyloxycarbonyl-l-proline: an in vitro and in vivo inhibitor of prolidase Biochim.. .Human recombinant prolidase from CHO and E coli A Lupi et al The thermostability of tagged and untagged recombinant prolidase expressed in E coli was evaluated: (a) by incubating the enzyme preactivated with 1 mm MnCl2 and 0.75 mm GSH for up to 6 days at 37 °C; (b) by adding to the incubation mixture 0.75 mm GSH or (c) 0.75 mm GSH and 1 mm MnCl2; (d) by supplementing... manganese-activated prolidase in control and prolidase- deficient cultured skin fibroblasts J Inherit Metab Dis 7, 32–34 38 Nakayama K, Awata S, Zhang J, Kaba H, Manabe M & Kodama H (2003) Characteristics of prolidase from the erythrocytes of normal humans and patients with prolidase deficiency and their mother Clin Chem Lab Med 41, 1323–1328 5478 39 Butterworth J & Priestman DA (1985) Presence in human cells and tissues... the same family J Med Genet, in press Fernandez-Espla MD, Martin-Hernandez MC & Fox PF (1997) Purification and characterization of a prolidase from Lactobacillus casei subsp casei IFPL 731 Appl Environ Microbiol 63, 314–316 Suga K, Kabashima T, Ito K, Tsuru D, Okamura H, Kataoka J & Yoshimoto T (1995) Prolidase from Xanthomonas maltophilia: purification and characterization of the enzyme Biosci Biotechnol... & Matsuda I (1989) Primary structure and gene localization of human prolidase J Biol Chem 264, 4476– 4481 15 Wang H, Kurien BT, Lundgren D, Patel NC, Kaufman KM, Miller DL, Porter AC, D’Souza A, Nye L, Tumbush J, et al (2006) A nonsense mutation of PEPD in Human recombinant prolidase from CHO and E coli 16 17 18 19 20 21 22 23 24 25 26 27 four Amish children with prolidase deficiency Am J Med Genet A... peroxide labelled anti-mouse IgG (Amersham Biosciences) at 1 : 5000 dilution ECL Plus (for CHO prolidase) and ECL (for E coli prolidase) reagents (Amersham Biosciences) were used for detection Identification of recombinant human prolidase The N-terminal sequence of E coli recombinant prolidase, both tagged and untagged, was determined by automated N-terminal Edman degradation on a Hewlett–Packard (Milan,... Biochem 59, 2087– 2090 Ghosh M, Grunden AM, Dunn DM, Weiss R & Adams MW (1998) Characterization of native and recombinant forms of an unusual cobalt-dependent proline dipeptidase (prolidase) from the hyperthermophilic archaeon Pyrococcus furiosus J Bacteriol 180, 4781–4789 Myara I, Charpentier C & Lemonnier A (1984) Prolidase and prolidase deficiency Life Sci 34, 1985–1998 Donald SP, Sun XY, Hu CA, Yu J,... obtained from a patient with PD and thus lacking endogenous prolidase activity (1 : 2.5) Prolidase activity was determined as described above from day 0 to day 6 Protein determination Protein concentration was measured by the Lowry method [46] using BSA as standard SDS ⁄ PAGE and western blotting analysis SDS ⁄ PAGE was performed in 10% acrylamide gels by the method of Laemmli under denaturing and reducing... Forlino A (2004) Characterization of a new PEPD allele causing prolidase deficiency in two unrelated patients: natural-occurrent mutations as a tool to investigate structure-function relationship J Hum Genet 49, 500–506 11 Ohhashi T, Ohno T, Arata J, Sugahara K & Kodama H (1990) Characterization of prolidase I and II from erythrocytes of a control, a patient with prolidase deficiency and her mother Clin... Clin Chem Lab Med 41, 1323–1328 5478 39 Butterworth J & Priestman DA (1985) Presence in human cells and tissues of two prolidases and their alteration in prolidase deficiency J Inherit Metab Dis 8, 193–197 40 Endo F, Matsuda I, Ogata A & Tanaka S (1982) Human erythrocyte prolidase and prolidase deficiency Pediatr Res 16, 227–231 41 Genta I, Perugini P, Pavanetto F, Maculotti K, Modena T, Casado B, Lupi . Human recombinant prolidase from eukaryotic and prokaryotic sources Expression, purification, characterization and long-term stability studies Anna. PD. Results Recombinant human prolidase expression and purification from eukaryotic and prokaryotic sources Total cellular RNA from cultured normal human fibroblasts