High expression of human carboxypeptidase M in Pichia pastoris: Purification and partial characterization

Autor(es)

R.B. Craveiro ^[1] ; J.D. Ramalho ^[2] ; J.R. Chagas ^[3] ; P.H.M. Wang ^[4] ; D.E. Casarini ^[5] ; J.L. Pesquero ; R.C. Araújo ^[7] ; J.B. Pesquero ^[8]

Afiliação do(s) autor(es)

^[1] Universidade Federal de São Paulo. Escola Paulista de Medicina. Departamento de Biofísica

^[2] Universidade de Mogi das Cruzes - Brasil

^[3] Universidade de Mogi das Cruzes - Brasil

^[4] Universidade Federal de São Paulo. Escola Paulista de Medicina. Departamento de Nefrologia - Brasil

^[5] Universidade Federal de São Paulo. Escola Paulista de Medicina. Departamento de Nefrologia - Brasil

^[7] Universidade de Mogi das Cruzes - Brasil

^[8] Universidade Federal de São Paulo. Escola Paulista de Medicina. Departamento de Biofísica

• Tipo de documento: Artigo Científico
• Fonte: Brazilian Journal of Medical and Biological Research; v. 39, n. 2, p. 211-217, 2006-02-00.

Resumo

Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidyl-inositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. The present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. The cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. The cpm gene, after cloning and transfection, was integrated into the yeast genome, which produced the active protein. The recombinant 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 filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein-1 min-1). This purification 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 (AU)

• Processo FAPESP: 00/03850-5 - Importância do sistema calicreína-cininas no desenvolvimento corporal: lições dos animais nocautes para o receptor B1 das cininas
• Beneficiário: Ronaldo de Carvalho Araújo
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 01/07538-9 - Projeto Genoma Estrutural
• Beneficiário: Ronaldo de Carvalho Araújo
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 01/03409-0 - Desenvolvimento corporal dos camundongos nocautes para os receptores B1 e B2 de cininas
• Beneficiário: Ronaldo de Carvalho Araújo
• Modalidade de apoio: Auxílio à Pesquisa - Jovens Pesquisadores