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Expression of human carboxypeptidase M in Pichia pastorisBrazilian Journal of Medical and Biological Research (2006) 39: 211-217ISSN 0100-879X

High expression of humancarboxypeptidase M in Pichia pastoris.Purification and partial characterization

1Departamento de Biofísica, 2Departamento de Nefrologia,Escola Paulista de Medicina, Universidade Federal de São Paulo,São Paulo, SP, Brasil3Universidade de Mogi das Cruzes, São Paulo, SP, Brasil4Departamento de Fisiologia e Biofísica, Universidade Federal de Minas Gerais,Belo Horizonte, MG, Brasil

R.B. Craveiro1,J.D. Ramalho3,

J.R. Chagas3,P.H.M. Wang2,D.E. Casarini2,

J.L. Pesquero3,4,R.C. Araújo3

and J.B. Pesquero1

Abstract

Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidyl-inositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and pro-teins at neutral pH. CPM plays an important role in the control ofpeptide hormones and growth factor activity on the cell surface. Thepresent study was carried out to clone and express human CPM in theyeast Pichia pastoris in order to evaluate the importance of thisenzyme in physiological and pathological processes. The cDNA forthe enzyme was amplified from total placental RNA by RT-PCR andcloned in the vector pPIC9, which uses the methanol oxidase promoterand drives the expression of high levels of heterologous proteins in P.pastoris. The cpm gene, after cloning and transfection, was integratedinto the yeast genome, which produced the active protein. The recom-binant protein was secreted into the medium and the enzymaticactivity was measured using the fluorescent substrate dansyl-Ala-Arg.The enzyme was purified by a two-step protocol including gel filtra-tion and ion-exchange chromatography, resulting in a 1753-foldpurified active protein (16474 RFU mg protein-1 min-1). This purifica-tion protocol permitted us to obtain 410 mg of the purified protein perliter of fermentation medium. SDS-PAGE showed that recombinantCPM migrated as a single band with a molecular mass similar to thatof native placental enzyme (62 kDa), suggesting that the expression ofa glycosylated protein had occurred. These results demonstrate for thefirst time the establishment of a method using P. pastoris to expresshuman CPM necessary to the development of specific antibodies andantagonists, and the analysis of the involvement of this peptidase indifferent physiological and pathological processes

CorrespondenceJ.B. Pesquero

Departamento de Biofísica

EPM, UNIFESP

Rua Botucatu, 862, 7º andar

04023-062 São Paulo, SP

Brasil

Fax: +55-11-5571-5780

E-mail: jbpesq@biofis.epm.br

Research supported by FAPESP

(Nos. 00/03850-5, 01/03409-0,

01/07538-9) and CNPq (No.

52.0012/02). R.B. Craveiro has

a master degree fellowship from

FAPESP (No. 02/04961-0).

Received June 14, 2005

Accepted October 27, 2005

Key words• Kinins• Human carboxypeptidase M• Recombinant protein• Pichia pastoris

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Introduction

Carboxypeptidase M (CPM) is an extra-cellular glycosylphosphatidyl-inositol-an-chored membrane glycoprotein. This pro-tein is a member of the CPN/E subfamily ofzinc metallo-carboxypeptidase. It specificallyremoves C-terminal basic residues such aslysine and arginine from peptides and fromproteins at neutral pH (1-4). The highestlevels of CPM have been found in humanlung and placenta, but significant amountsare present in kidney, blood vessels, intes-tine, brain, and peripheral nerves (1,5-7).CPM has been found also in soluble form invarious body fluids, including amniotic fluid,seminal plasma and urine (5,8,9). Due to itswide distribution in a variety of tissues, it isbelieved to play important roles in the con-trol of peptide hormone and growth factoractivity on the cell surface, and in the mem-brane-localized degradation of extracellularproteins (10).

By removing the C-terminal Arg residueof the intact kinins (and related peptides),CPM can switch the receptor specificity (11),inactivating the agonist for the B2 receptorand concomitantly producing an agonist forthe B1 receptor (1). Recent results from ourgroup have shown that the kinin B1 receptoris important for neutrophil migration intoinflamed tissues (12,13) and for glucose/insulin homeostasis (Araujo RC, unpublisheddata). Furthermore, CPM is recognized byantibodies that detect cell surface antigenson macrophages and is strongly induced inthe differentiation of monocytes to macro-phages (14). Therefore, the mechanism forneutrophil migration could be dependent onthe CPM present in the cell membrane ofmacrophages, indicating a relevant role ofthis enzyme at sites of inflammation. In ad-dition, the conversion of agonistic activitywas shown to result in an enhanced andprolonged output of nitric oxide in responseto bradykinin or kallidin in cytokine-stimu-lated human lung microvascular endothelial

cells (15). By cloning the cDNA from differ-ent human tissues, we could determine themolecular structure (16) and show the pres-ence of alternatively spliced messages forCPM. Furthermore, we could also show thatthe expression of this carboxypeptidase intumor tissues is differently regulated, rais-ing the question of the role of this enzyme incancer (16). Thus, taking into account thefunction played by kinins and nitric oxide ininflammatory diseases and cancer (11), andthe fact that specific inhibitors of this car-boxypeptidase are still lacking, CPM mightbe an important target for the developmentof new drugs to treat these pathologies.

Recently, a human CPM (hCPM) wasexpressed by using a system based on bacu-lovirus-infected insect cells (Sf9) (17). Thecited study aimed to identify the membrane-anchor signal and the glycosylphosphatidyl-inositol attachment site and to investigatethe roles of the two conserved glutamic acidresidues, Glu260 and Glu264, in kinetic param-eters and in protein stability. Furthermore,the same group determined the crystal struc-ture of the human enzyme (18).

Therefore, the objective of the presentstudy was to obtain an active glycosylatedprotein using the expression system of theyeast Pichia pastoris. This system has sev-eral advantages, including the use of thealcohol oxidase I gene promoter, the abilityto stably integrate expression plasmids atspecific sites, the ability of the cells to becultivated at high density, and a simplifiedpurification procedure for secreted heterolo-gous proteins. Moreover, similar to mam-malian and insect cells, P. pastoris can carryout certain co- and post-translational modi-fications of foreign proteins (19,20). Theresults described in the present report dem-onstrate a high level of expression of theactive recombinant hCPM with a molecularmass similar to the native glycoprotein (62kDa), indicating that the P. pastoris expres-sion system can be useful for the productionof active CPM at relevant levels.

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Material and Methods

Enzymes, primers and vectors for molec-ular biology procedures were purchased fromInvitrogen (Carlsbad, CA, USA) and Pro-mega (Madison, WI, USA). The dansyl-Ala-Arg substrate was from Bachem (King ofPrussia, PA, USA). DNA sequencing wasperformed using an ABI377 sequencer (Ap-plied Biosystems, Foster City, CA, USA).The Amicon Ultra 30,000 MWCO mem-brane was from Millipore (Billerica, MA,USA), and the Superdex 200 HR 10/30 andMono Q HR 5/5 columns were from Amer-sham Biosciences (Piscataway, NJ, USA).

RNA extraction and reverse transcriptionpolymerase chain reaction

Total RNA from human placenta wasisolated using the Trizol reagent accordingto the manufacturer’s protocol and its puritywas evaluated by electrophoresis on 1% aga-rose gel. Contamination of RNA sampleswith genomic DNA was avoided by treat-ment for 1 h at 37ºC with 1 U RNAse-free/DNAse I per 2 µg RNA. For the same amountof RNA, the reaction also contained 20 URnaseOUT-Rnase inhibitor and 3 mMMgCl2. After incubation, samples wereheated to 95ºC and immediately chilled onice for DNAse I denaturation. Reverse trans-cription was performed using 2 µg total RNA,200 U Moloney murine leukemia virus IIreverse transcriptase, 5 mM DTT, 50 ngrandom hexamer primers, 1X PCR buffer,0.5 mM dNTPs, and 3 mM MgCl2. Reac-tions were carried out at 20ºC for 10 min,42ºC for 45 min, 95ºC for 5 min, and 4ºC for10 min. The resulting cDNA from the hu-man placenta was then used for PCR whichwas performed with 500 ng cDNA. The PCRmixture contained 0.5 µM concentrations ofsense (hCPM5 5'-GAATTCTTGCTGCCTTTGGTAGCTGC) and antisense (CPMAS25'-GCGGCCGCTTATTTGAAGAATATGTGC) primers, which contained added sites

for the restriction enzymes EcoRI and NotI,respectively, 2 U platinum TaqDNA poly-merase, 1.5 mM MgCl2, 50 mM KCl, and 20mM Tris-HCl, pH 8.3, in a 50-µL volume.PCR amplification was performed by incu-bating the samples at 94ºC for 1 min, fol-lowed by 30 cycles at 94ºC for 1 min, and at60ºC for 90 s, with a final extension at 72ºCfor 7 min. At the end of amplification,samples were submitted to electrophoresison 1% agarose gel with a 100-bp DNA lad-der as a size marker. The amplified bands of1313 bp were visualized by ethidium bro-mide staining.

Construction of expression vector pPIC9-cpm

The PCR-amplified fragment encodinghCPM was cloned into the pGEM-T-Easy(Promega) cloning vector and subjected todouble-stranded DNA sequencing (ABI 377,Applied Biosystems) After EcoRI-NotI di-gestion, the cpm gene was cloned in frameinto the pPIC9 (Invitrogen) vector betweenthe EcoRI (5' end) and NotI (3' end) restric-tion sites to generate the plasmid pPIC9-cpm.

Transformation of Pichia pastoris andselection of high-level expression colonies

The following culture media used fortransformation of P. pastoris, selection ofrecombinant clones, and expression of CPMwere prepared according to manufacturerrecommendations (Invitrogen): minimal dex-trose medium (MD), minimal methanol me-dium (MM), buffered glycerol-complexmedium (BMGY), and buffered minimalmethanol (BMMY). For cultures in liquidBMMY, which contain methanol as an in-ducer and carbon source, methanol was addedevery 24 h to a final concentration of 0.5%(v/v). All cultures were carried out at 30oC.The pPIC9-cpm construct (10 µg) was lin-earized with BglII and transformed into P.pastoris wild-type strain GS115 (Invitrogen)

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by electroporation at 25 µF, 400 Ω, and 1500V using a Bio-Rad (Hercules, CA, USA)GenePulser. Immediately after pulsing, 1mL cold 1 M sorbitol was added to the P.pastoris cells, and plated onto MD contain-ing 1 M sorbitol. After cultivation, all trans-formants were patched or replica plated ontoboth MM and MD plates to select the colo-nies growing more slowly in MM than inMD, and these colonies were cultivated inliquid BMMY. To analyze cpm gene inte-gration into the P. pastoris genome, genom-ic DNA was isolated from different clonesand integration was confirmed by PCR us-ing the same cloning primers. CPM activityof positive clones in the BMMY supernatantwas measured with the dansyl-Ala-Arg sub-strate as previously described (21). The clonepresenting the highest CPM activity wasinoculated into 100 mL BMGY and culturedfor 1 day. The cells were then collected bycentrifugation and resuspended in 500 mLBMMY. Every day (0, 24, 48, 72, and 96 h),just before the addition of 0.5% methanol,an aliquot of the medium was collected tomeasure CPM activity and protein concen-tration.

Purification of recombinantcarboxypeptidase M

The CPM secreted in the supernatant waspurified by a two-step procedure consistingof gel filtration and ion-exchange chroma-tography. Briefly, the expression mediumwas concentrated on an Amicon concentra-tor with an Amicon Ultra 30,000 MWCOmembrane (Millipore), and loaded on aSuperdex 200 HR 10/30 column (Amer-sham Biosciences) equilibrated with 50 mMNa-phosphate buffer, pH 7, containing 0.15M NaCl. The column was eluted with theequilibration buffer at a flow rate 0.8 mL/min, and 0.5-mL fractions were collectedand assayed for CPM activity. The effluentfrom the gel filtration column containingenzyme activity was combined and loaded

onto a Mono Q HR 5/5 column (AmershamBiosciences) equilibrated with 50 mM Tris-HCl buffer, pH 8. The column was washedwith equilibration buffer and then elutedwith a linear gradient of 0 to 1 M NaCl in thesame buffer. Proteins were detected by UVabsorption at 280 nm, 0.5-mL fractions werecollected and assayed for CPM activity.

Protein concentration was determined bythe dye-binding method of Bradford (22)using bovine serum albumin as a standard.

Results and Discussion

Analysis of transformed clones andexpression of human carboxypeptidase M

Total RNA from human placenta wasisolated and submitted to the reverse trans-cription. The resultant cDNA was then usedfor PCR and yielded a 1313-bp DNA frag-ment containing the whole coding regionwith the expected sequence, except for onebase change at position 1158 (T → A; datanot shown). However, this base alterationdoes not lead to a change in the amino acidsequence of the generated protein. After di-gestion with EcoRI-NotI restriction enzymes,the cpm gene was cloned in frame in thepPIC9. The generated plasmid pPIC9-cpmcontaining the hCPM gene was transformedinto the P. pastoris wild-type strain GS115.Some of the colonies selected on MD plates(10 of 34 colonies) were tested by PCR toconfirm the integration of the CPM codingregion into the P. pastoris genome and sixpositive clones were selected. The clone withthe highest activity (9.4 RFU min-1 mg pro-tein-1) and one negative control, pPIC9 (with-out the insert), were initially inoculated intoBMGY and later into BMMY. The cells andthe supernatant of these clones were thencollected by centrifugation, CPM activitywas measured and protein concentration wasdetermined. As shown in Figure 1, carbox-ypeptidase activity was detected at high lev-els in the supernatant of the positive clone.

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Figure 2 shows the levels of the total carbox-ypeptidase activity at different times. Asobserved, CPM activity increased in themedium in a time-dependent manner. Theadvantage of expressing heterologous pro-teins as secreted proteins is that P. pastorissecretes very low levels of native proteins.This, combined with the very low amount ofprotein in the minimal Pichia growth medi-um, means that the medium serves as thefirst step in the purification of the protein(19).

Purification of human carboxypeptidase M

The supernatant was concentratedthrough an Amicon membrane and submit-ted to gel filtration chromatography (Super-dex 200 HR 10/30). Protein elution wasmonitored by measuring the absorbance at280 nm and the carboxypeptidase activitywas evaluated with the substrate dansyl-Ala-Arg. Protein eluted from the columnshowed one major peak of activity with amolecular mass estimated between 40 and90 kDa (Figure 3A). The fractions corre-sponding to this major peak were pooled (P)and submitted to SDS-PAGE. The resultsshowed the presence of three main bands inthe gel (Figure 3B). The active fraction wassubmitted to ion-exchange chromatography(Mono Q 5/5) which resulted in only onepeak of activity and a band in SDS-PAGEaround 62-70 kDa (Figure 4), similar to the62 kDa corresponding to the native placentalprotein. This small difference in molecularmass could be attributed to the partial cleav-age produced by the P. pastoris STE13 pro-tease in the recombinant protein, leavingfour additional amino acids (EAEA) at theN-terminal region (23). Another reason couldbe the differences in the number and type ofsugar units added to the yeast-secreted pro-teins compared with human proteins (24).

The purification steps resulted in 1753-fold purification of recombinant hCPM. Thespecific activity of the purified enzyme was

Figure 1. Expression of recom-binant hCPM in the control andtransformed (hCPM) clones ofPichia pastoris 24 h after inocu-lation into buffered glycerol-complex medium. The yeastcells (y) and the supernatant (s)were separated by centrifuga-tion and assayed for CPM activ-ity with dansil-Ala-Arg as sub-strate. hCPM = human carbox-ypeptidase M; RFU = relative flu-orescence units. *P < 0.01 com-pared to the supernatant of thecontrol clone (t-test).

Figure 2. Carboxypeptidase Mactivity in the culture superna-tant of the GS115-CPM strain.Cells were induced in bufferedminimal methanol and the activ-ity was evaluated with the sub-strate dansil-Ala-Arg at differenttimes. RFU = relative fluores-cence units. *P < 0.05 and **P <0.01 compared to the 24-h cul-ture (t-test).

Figure 3. A, Gel filtration of recombinant human carboxypeptidase M (CPM). The concen-trated supernatant (3.05 mg/mL in 2 mL) was loaded onto a Superdex 200 HR 10/30 columnin the FPLC system. The column was equilibrated with 50 mM Na-phosphate buffer, pH 7,containing 0.15 M NaCl and eluted with the equilibration buffer at a flow rate of 0.8 mL/min.Fractions of 0.5 mL were collected and assayed for CPM activity. RFU = relative fluores-cence units. B, SDS-PAGE (12%) of pooled fractions containing CPM activity (P). M,molecular mass markers (Bench Mark Protein Ladder, Invitrogen).

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16474 RFU mg-1 min-1 using dansyl-Ala-Arg as substrate (Table 1). The very lowlevels of specific activity observed for thecrude supernatant when compared to thepurified enzyme may be explained by thepresence of inhibitors of the enzyme in thefermentation medium. Some salts added tothe medium at high concentrations are ableto inhibit CPM and probably are separatedfrom the enzyme during the purification pro-cess, causing a large increase in specific andtotal activities.

The latest achievements in the expressionsystem of P. pastoris have changed deeply themethodology used to express heterologous

proteins, increasing the expression levels andbioactivity of the latter. In addition, the greatsuccess of P. pastoris is related to the fact thatthis organism is a eukaryote, capable to ex-press proteins with post-translational modifi-cations, sometimes essential for proper func-tion. A further benefit of the P. pastoris systemis that strong promoters are available to drivethe expression of the foreign gene(s), thusenabling production of large amounts of thetarget protein(s) with relative technical easeand at a lower cost than most other eukaryoticsystems (25).

The results shown here demonstrate theestablishment of a method using P. pastoristo express CPM in high yield of purifiedenzyme, i.e., 410 mg/L of fermentation cul-ture. The expression levels of the hCPM inPichia obtained in the present study wasmuch higher (around 10-fold) when com-pared to the published expression systemusing baculovirus-infected insect cells, about30-40 mg/L of recombinant mutant CPM inthe culture medium (17). Some investiga-tors, using the same system, have also de-scribed a similar recovery for the enzymetannin acyl hydrolase (25), and even higherrecoveries for the enzyme lipase/acyltrans-ferase from Candida parapsilosis (EC 3.1.1.3)(26), and for the tetanus toxin fragment C(24,27), highlighting the potential of thistechnique to express high yield of heterolo-gous proteins.

The present data show that we developed amethodology that permits the production oflarge amounts of the CPM enzyme, necessaryfor the development of specific antibodies andantagonists and for the analysis of the involve-ment of this peptidase in different physiologi-cal and pathological processes.

Acknowledgments

We thank Nelson Mora and Ivan Cordeirofrom the Departamento de Biofísica da Uni-versidade Federal de São Paulo, São Paulo,SP, Brazil, for excellent technical assistance.

Figure 4. A, Ion-exchange chromatography. A pool of effluent containing enzyme activity(0.28 mg/mL in 1.5 mL) obtained by gel filtration (Figure 3) was loaded onto a Mono Q HR 5/5 column equilibrated with 50 mM Tris-HCl buffer, pH 8. The column was washed withequilibration buffer and then eluted with a linear gradient (0 to 1 M) NaCl in the same buffer.Proteins were detected by UV absorption at 280 nm. Fractions of 0.5 mL were collected andassayed for carboxypeptidase M activity. RFU = relative fluorescence units. B, SDS-PAGE(12%) of pooled active fractions. M, molecular mass markers (Bench Mark Protein Ladder,Invitrogen). Human carboxypeptidase M (hCPM) appears as a diffuse band because it is aglycoprotein.

Table 1. Purification of recombinant hCPM from 1 mL of fermentation supernatant.

Step Protein Total Specific Purification(mg) activity* activity** (X-fold)

Supernatant 6.10 57.3 9.4 1Superdex 200 HR 0.86 2077.0 2415 257Mono Q 0.41 6754.3 16474 1753

*RFU/min. **RFU min-1 mg protein-1. RFU = relative fluorescence units; hCPM =human carboxypeptidase M. Purification = Mono Q specific activity/supernatant spe-cific activity.

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