High Expression of Human Carboxypeptidase M in Pichia Pastoris
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Brazilian Journal of Medical and Biological Research (2006) 39: 211-217 Expression of human carboxypeptidase M in Pichia pastoris 211 ISSN 0100-879X High expression of human carboxypeptidase M in Pichia pastoris. Purification and partial characterization R.B. Craveiro1, 1Departamento de Biofísica, 2Departamento de Nefrologia, J.D. Ramalho3, Escola Paulista de Medicina, Universidade Federal de São Paulo, J.R. Chagas3, São Paulo, SP, Brasil P.H.M. Wang2, 3Universidade de Mogi das Cruzes, São Paulo, SP, Brasil D.E. Casarini2, 4Departamento de Fisiologia e Biofísica, Universidade Federal de Minas Gerais, J.L. Pesquero3,4, Belo Horizonte, MG, Brasil R.C. Araújo3 and J.B. Pesquero1 Abstract Correspondence Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidyl- Key words J.B. Pesquero inositol-anchored membrane glycoprotein, which removes the C- • Kinins Departamento de Biofísica terminal basic residues, lysine and arginine, from peptides and pro- • Human carboxypeptidase M EPM, UNIFESP teins at neutral pH. CPM plays an important role in the control of • Recombinant protein Rua Botucatu, 862, 7º andar peptide hormones and growth factor activity on the cell surface. The • Pichia pastoris 04023-062 São Paulo, SP Brasil present study was carried out to clone and express human CPM in the Fax: +55-11-5571-5780 yeast Pichia pastoris in order to evaluate the importance of this E-mail: [email protected] enzyme in physiological and pathological processes. The cDNA for the enzyme was amplified from total placental RNA by RT-PCR and Research supported by FAPESP cloned in the vector pPIC9, which uses the methanol oxidase promoter (Nos. 00/03850-5, 01/03409-0, and drives the expression of high levels of heterologous proteins in P. 01/07538-9) and CNPq (No. pastoris. The cpm gene, after cloning and transfection, was integrated 52.0012/02). R.B. Craveiro has a master degree fellowship from into the yeast genome, which produced the active protein. The recom- FAPESP (No. 02/04961-0). binant protein was secreted into the medium and the enzymatic activity 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-fold Received June 14, 2005 purified active protein (16474 RFU mg protein-1 min-1). This purifica- Accepted October 27, 2005 tion protocol permitted us to obtain 410 mg of the purified protein per liter of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa), suggesting that the expression of a glycosylated protein had occurred. These results demonstrate for the first time the establishment of a method using P. pastoris to express human CPM necessary to the development of specific antibodies and antagonists, and the analysis of the involvement of this peptidase in different physiological and pathological processes Braz J Med Biol Res 39(2) 2006 212 R.B. Craveiro et al. Introduction cells (15). By cloning the cDNA from differ- ent human tissues, we could determine the Carboxypeptidase M (CPM) is an extra- molecular structure (16) and show the pres- cellular glycosylphosphatidyl-inositol-an- ence of alternatively spliced messages for chored membrane glycoprotein. This pro- CPM. Furthermore, we could also show that tein is a member of the CPN/E subfamily of the expression of this carboxypeptidase in zinc metallo-carboxypeptidase. It specifically tumor tissues is differently regulated, rais- removes C-terminal basic residues such as ing the question of the role of this enzyme in lysine and arginine from peptides and from cancer (16). Thus, taking into account the proteins at neutral pH (1-4). The highest function played by kinins and nitric oxide in levels of CPM have been found in human inflammatory diseases and cancer (11), and lung and placenta, but significant amounts the fact that specific inhibitors of this car- are present in kidney, blood vessels, intes- boxypeptidase are still lacking, CPM might tine, brain, and peripheral nerves (1,5-7). be an important target for the development CPM has been found also in soluble form in of new drugs to treat these pathologies. various body fluids, including amniotic fluid, Recently, a human CPM (hCPM) was seminal plasma and urine (5,8,9). Due to its expressed by using a system based on bacu- wide distribution in a variety of tissues, it is lovirus-infected insect cells (Sf9) (17). The believed to play important roles in the con- cited study aimed to identify the membrane- trol of peptide hormone and growth factor anchor signal and the glycosylphosphatidyl- activity on the cell surface, and in the mem- inositol attachment site and to investigate brane-localized degradation of extracellular the roles of the two conserved glutamic acid proteins (10). residues, Glu260 and Glu264, in kinetic param- By removing the C-terminal Arg residue eters and in protein stability. Furthermore, of the intact kinins (and related peptides), the same group determined the crystal struc- CPM can switch the receptor specificity (11), ture of the human enzyme (18). inactivating the agonist for the B2 receptor Therefore, the objective of the present and concomitantly producing an agonist for study was to obtain an active glycosylated the B1 receptor (1). Recent results from our protein using the expression system of the group have shown that the kinin B1 receptor yeast Pichia pastoris. This system has sev- is important for neutrophil migration into eral advantages, including the use of the inflamed tissues (12,13) and for glucose/ alcohol oxidase I gene promoter, the ability insulin homeostasis (Araujo RC, unpublished to stably integrate expression plasmids at data). Furthermore, CPM is recognized by specific sites, the ability of the cells to be antibodies that detect cell surface antigens cultivated at high density, and a simplified on macrophages and is strongly induced in purification procedure for secreted heterolo- the differentiation of monocytes to macro- gous proteins. Moreover, similar to mam- phages (14). Therefore, the mechanism for malian and insect cells, P. pastoris can carry neutrophil migration could be dependent on out certain co- and post-translational modi- the CPM present in the cell membrane of fications of foreign proteins (19,20). The macrophages, indicating a relevant role of results described in the present report dem- this enzyme at sites of inflammation. In ad- onstrate a high level of expression of the dition, the conversion of agonistic activity active recombinant hCPM with a molecular was shown to result in an enhanced and mass similar to the native glycoprotein (62 prolonged output of nitric oxide in response kDa), indicating that the P. pastoris expres- to bradykinin or kallidin in cytokine-stimu- sion system can be useful for the production lated human lung microvascular endothelial of active CPM at relevant levels. Braz J Med Biol Res 39(2) 2006 Expression of human carboxypeptidase M in Pichia pastoris 213 Material and Methods for the restriction enzymes EcoRI and NotI, respectively, 2 U platinum TaqDNA poly- Enzymes, primers and vectors for molec- merase, 1.5 mM MgCl2, 50 mM KCl, and 20 ular biology procedures were purchased from mM Tris-HCl, pH 8.3, in a 50-µL volume. Invitrogen (Carlsbad, CA, USA) and Pro- PCR amplification was performed by incu- mega (Madison, WI, USA). The dansyl-Ala- bating the samples at 94ºC for 1 min, fol- Arg substrate was from Bachem (King of lowed by 30 cycles at 94ºC for 1 min, and at Prussia, PA, USA). DNA sequencing was 60ºC for 90 s, with a final extension at 72ºC performed using an ABI377 sequencer (Ap- for 7 min. At the end of amplification, plied Biosystems, Foster City, CA, USA). samples were submitted to electrophoresis The Amicon Ultra 30,000 MWCO mem- on 1% agarose gel with a 100-bp DNA lad- brane was from Millipore (Billerica, MA, der as a size marker. The amplified bands of USA), and the Superdex 200 HR 10/30 and 1313 bp were visualized by ethidium bro- Mono Q HR 5/5 columns were from Amer- mide staining. sham Biosciences (Piscataway, NJ, USA). Construction of expression vector pPIC9-cpm RNA extraction and reverse transcription polymerase chain reaction The PCR-amplified fragment encoding hCPM was cloned into the pGEM-T-Easy Total RNA from human placenta was (Promega) cloning vector and subjected to isolated using the Trizol reagent according double-stranded DNA sequencing (ABI 377, to the manufacturer’s protocol and its purity Applied Biosystems) After EcoRI-NotI di- was evaluated by electrophoresis on 1% aga- gestion, the cpm gene was cloned in frame rose gel. Contamination of RNA samples into the pPIC9 (Invitrogen) vector between with genomic DNA was avoided by treat- the EcoRI (5' end) and NotI (3' end) restric- ment for 1 h at 37ºC with 1 U RNAse-free/ tion sites to generate the plasmid pPIC9- DNAse I per 2 µg RNA. For the same amount cpm. of RNA, the reaction also contained 20 U RnaseOUT-Rnase inhibitor and 3 mM Transformation of Pichia pastoris and MgCl2. After incubation, samples were selection of high-level expression colonies heated to 95ºC and immediately chilled on ice for DNAse I denaturation. Reverse trans- The following culture media used for cription was performed using 2 µg total RNA, transformation of P. pastoris, selection of 200 U Moloney murine leukemia virus II recombinant clones, and expression of CPM reverse transcriptase, 5 mM DTT, 50 ng were prepared according to manufacturer random hexamer primers, 1X PCR buffer, recommendations (Invitrogen): minimal dex- 0.5 mM dNTPs, and 3 mM MgCl2. Reac- trose medium (MD), minimal methanol me- tions were carried out at 20ºC for 10 min, dium (MM), buffered glycerol-complex 42ºC for 45 min, 95ºC for 5 min, and 4ºC for medium (BMGY), and buffered minimal 10 min.