<a name="home"></a>High expression of human carboxypeptidase M in Pichia pastoris: Purification and partial characterization

Abstract

Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidylinositol-anchored membrane glycoprotein, which removes the Cterminal 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

Identification of Common Pathways Mediating Differentiation of Bone Marrow- and Adipose Tissue-Derived Human Mesenchymal Stem Cells into Three Mesenchymal Lineages

“…Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidylinositol‐anchored membrane glycoprotein. It plays an important role in the control of peptide hormones, growth factor activity at the cell surface, and the membrane‐localized degradation of extracellular proteins [26].…”

Production of L1 protein from different types of HPV in Pichia pastoris using an integrative vector

“…However, according to Higgins and Cregg (28), there is a 50-75% probability that the P. pastoris system will produce foreign proteins at a reasonable level without the need of optimization. Since 1984 different proteins have been well expressed in P. pastoris and several studies have demonstrated the strong and intrinsic Pichia ability to express heterologous genes without optimizations (14,29,30). Bazan et al (18) demonstrated that, using the episomal vector, the production of the HPV-16 L1 protein in P. pastoris was only possible after codon optimization of the L1 gene.…”

Pichia pastoris: A Recombinant Microfactory for Antibodies and Human Membrane Proteins

“…In fact, the application of affinity chromatography as the main tool for MP capture from total crude extract or total membrane fraction remains one of the most common and successfully employed techniques for the first step of MP purification. However, molecular exclusion and ion-exchange chromatographies have also been successfully employed [18].…”

“…Additionally, as Pichia does not secrete high levels of native proteins, the purification of secreted formulations is much more simple than in other systems [73]. Moreover, the simplicity of the techniques needed for molecular genetic manipulation and their similarity to those used with Saccharomyces cerevisiae have made Pichia one of the most well-characterized experimental systems in modern biology [18].…”