Báo cáo khoa học: Human recombinant prolidase from eukaryotic and prokaryotic sources Expression, purification, characterization and long-term stability studies pptx

[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 defic

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 Mn2+-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 37C 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 Mn2+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).

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 37C 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· 106 stable transfected CHO cells were plated in T175 flasks and harvested after 24, 48, 96 and 120 h at

37C and 5% CO2 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)1ÆmL)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,

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) ⁄ 4 mm

2-mercaptoethanol at 4C 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 4C 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

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.

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 35C 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

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-(Gly-Pro > Phe-(Gly-Pro > Leu-(Gly-Pro) (Table 1) Cbz-Pro is a well known in vitro and in vivo human prolidase inhibitor [29] Activity studies confirmed

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 m M 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 (%)

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 60C (at 20, 37, 50, 70, 80 C), with

the optimum activity at 50C (the activity at this

tem-perature was assumed to be 100%) At 37C, the

activity of the recombinant His-tagged enzyme was

50% of the maximum activity measured at 50C, 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 (Ca2+, Co2+,

Mg2+, Mn2+and Zn2+) 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 MnCl2 preactivation The maximum activity

was obtained with Mn2+ (assumed to be 100%) Less

than 30% of the activity was obtained with the other

metals tested (Fig 4C)

Long-term stability at 37C

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 37C 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 50C in presence of

MnCl2 and GSH and incubated at 37C 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, P¼ 0.103) (Fig 5A)

To improve enzyme activity over time, we incubated the preactivated recombinant enzymes at 37C in pres-ence of GSH or of GSH and MnCl2 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 MnCl2 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, P¼ 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 Mn2+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

37C 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 (%)

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 (37C in the presence of MnCl2 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 37C

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.

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)3mmolÆh)1Æmg)1 and 1.315 ± 0.525 mmolÆ

h)1Æmg)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 Mn2+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.

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

50C and a pH optimum of 7.8 However, at the

phy-siological temperature of 37C, 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 Mn2+ ions [33], and this metal cannot be

effectively replaced by other metals Similarly, our

recombinant enzyme showed highest activity in the

presence of MnCl2, with activities of less than 30% in

the presence of Ca2+, Co2+, Mg2+ and Zn2+

Fur-thermore, preactivation with Mn2+ before purification

by IMAC was a requirement for the enzyme

synthes-ized in CHO cells; no binding to the Ni2+was possible

without preactivation (data not shown) The amount

of purified recombinant prolidase from E coli was

increased by including MnCl2 in the culture medium

and preactivating the enzyme with Mn2+ 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 Mn2+ 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 37C

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 MnCl2, GSH and BSA in a biodegradable micro-sphere This system allowed greater release of the enzyme in vitro after 20 h of incubation at 37C, 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 37C in the presence of 5% CO2 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

Escherichia coli BL21 (DE3) [F– ompT hsdSB(rB–mB–)gal

dcm (DE3)] bacteria were grown in Luria-Bertani medium

in an orbital incubator at 37C 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 94C

for 2 min, 35 cycles at 94C for 30 s, 65 C for 30 s, and

68C 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 37C 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· 105 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 coliBL21 (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· 106 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⁄ Pi washes, the cell layer was mechanically removed Pelleted cells were suspended in

50 mm Tris⁄ HCl (pH 7.8) ⁄ 4 mm 2-mercaptoethanol, soni-cated on ice, and centrifuged at 16 000 g for 30 min at

4C The lysate was incubated with 0.75 mm GSH and

1 mm MnCl2 for 12 h at 4C, then dialysed overnight at

4C against 50 mm Tris ⁄ HCl (pH 7.8) ⁄ 4 mm 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 4C 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) ⁄ 4 mm 2-mercaptoethanol at 4 C The purified enzyme was stored at)80 C in 0.3-mL aliquots in LoBind tubes (Eppendorf, Milan, Italy)

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 MnCl2 at 37C in an orbital

incuba-tor and agitated at 190 r.p.m At A600¼ 0.6, the cells were

induced with 1 mm isopropyl b-d-1-thiogalactopyranoside

for 2 h at 37C

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 ⁄ 1 mm EDTA ⁄ 4 mm

2-mercaptoeth-anol⁄ 4 mm 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 4C Supernatants were filtered on

0.22-lm filters and diluted fivefold with 20 mm Tris⁄ HCl

(pH 7.8)⁄ 4 mm 2-mercaptoethanol The solution containing

the recombinant prolidase was preactivated by incubation

with GSH (0.75 mm) and MnCl2 (1 mm) for 1 h at 50C

The Mn2+ excess was eliminated by dialysing the sample

for 24 h at 4C against 20 mm Tris ⁄ HCl (pH 7.8) ⁄ 4 mm

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) ⁄ 4 mm 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· 105

in T75 tissue culture flasks and grown for 8 days in

DMEM After medium removal, the cell layer was washed

with NaCl⁄ Pi, mechanically removed in 50 mm Tris⁄ HCl

(pH 7.8)⁄ 4 mm 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 20C 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 35C 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) ⁄ 4 mm 2-mercaptoethanol was added with NaCl, CaCl2and imidazole to a final concentra-tion of 100 mm, 5 mm and 20 mm, respectively, and loaded

on the column in the presence of 4 UÆmg)1Factor Xa The column was incubated at 35C 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 MnCl2, 0.75 mm GSH and 100 mm gly-cyl-l-proline (Gly-Pro; MP Biomedicals, Milan, Italy) at

37C 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, 80C)

Metal ion dependence was determined by incubating with different bivalent ions [Ca2+, Co2+, Mg2+, Mn2+ and

Zn2+ (salt stock solution 100 mm)] the enzyme obtained from E coli without preactivation with Mn2+and fibroblast lysate