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

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

Summa rerum genera esse tantum duo: substantiam nimirum & accidens, ex suffragio ampliss. facult. philos. in florentissimo ad Auram Athenaeo, sub umbone ... dn. mag. Simonis Tålpo, met & log. professoris famigeratissimi, consistorii Acad. assessoris gravissimi nec non annexae Pijkensis pastoris longe fidelissimi. Summos in philosophia honores aditurus dissertatione philosophica ad sobrie philosophantium examen modeste delata statuere conabitur Jeremias Wallenius J. f. Tavastensis. Ad diem 21. Nov. anni redempti orbis M. DC. XC. IV. Horis pomeridianis in auditorio maximo

Invokaatio: A.J.

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

Human papillomavirus (HPV) infection is the most common sexually transmitted disease in the world and is related to the etiology of cervical cancer. The most common high-risk HPV types are 16 and 18; however, the second most prevalent type in the Midwestern region of Brazil is HPV-33. New vaccine strategies against HPV have shown that virus-like particles (VLP) of the major capsid protein (L1) induce efficient production of antibodies, which confer protection against the same viral type. The methylotrophic yeast Pichia pastoris is an efficient and inexpensive expression system for the production of high levels of heterologous proteins stably using a wild-type gene in combination with an integrative vector. It was recently demonstrated that P. pastoris can produce the HPV-16 L1 protein by using an episomal vector associated with the optimized L1 gene. However, the use of an episomal vector is not appropriate for protein production on an industrial scale. In the present study, the vectors were integrated into the Pichia genome and the results were positive for L1 gene transcription and protein production, both intracellularly and in the extracellular environment. Despite the great potential for expression by the P. pastoris system, our results suggest a low yield of L1 recombinant protein, which, however, does not make this system unworkable. The achievement of stable clones containing the expression cassettes integrated in the genome may permit optimizations that could enable the establishment of a platform for the production of VLP-based vaccines.

Biosíntesis de la hormona del crecimiento humano recombinante (HGHr) en Pichia pastoris y Escherichia coli

Tesis (Maestría en Ciencias con Especialidad en Biología Molecular e Ingeniería Genética) UANL

Construcción de cepas de pichia pastoris productoras de hormonas del crecimiento humana recombinante y evaluación de esquemas de purificación

Tesis (Maestría en Ciencias con Especialidad en Biología Molecular e Ingeniería Genética) UANL

Construcción de cepas de pichia pastoris portadoras del DNAc de la hormona del crecimiento bovino

Tesis (Maestría en Ciencias con Especialidad en Biología Molecular e Ingeniería Genética) UANL

Estudio molecular y celular bajo la influencia de condiciones de cultivo durante la producción de proteínas heterólogas en Pichia pastoris

Tesis (Maestría en Ciencias con Especialidad en Biología Molecular e Ingeniería Genética) UANL

Construcción de una cepa de Pichia pastoris sobreproductora de la isoforma de 20 kDa de la hormona del crecimiento humano

Tesis (Maestría en Ciencias con Especialidad en Biología Molecular e Ingeniería Genética) UANL

Hormona del crecimiento caprino : construcción de un DNA que la codifica y su expresión en Pichia pastoris

Tesis (Maestría en Ciencias con Especialidad en Biología Molecular e Ingeniería Genética) UANL

Clonación molecular del DNAc de la hormona variante del crecimiento humano y su expresión en Pichia pastoris

Tesis (Maestría en Ciencias con Especialidad en Biología Molecular e Ingeniería Genética) UANL

Purificación de la hormona del crecimiento humano recombinante producida en pichia pastoris

Tesis (Maestría en Ciencias con Especialidad en Biología Molecular e Ingeniería Genética) UANL

Desarrollo de un esquema de purificación para la hormona de crecimiento bovina producida por Pichia pastoris

Tesis (Maestría en Ciencias con Especialidad en Biología Molecular e Ingeniería Genética) U.A.N.L. Facultad de Medicina 2006.

Establecimiento de un método de purificación de proteínas recombinantes, basado en etiquetas de histidina, producidas en pichia pastoris.

Tesis (Maestro en Ciencias con Especialidad en Biología Molecular e Ingeniería Genética) UANL, 2011.

Construcción de Cepas recombinantes de pichia pastoris productoras de un tripsinógeno quimérico.

Tesis (Maestría en Ciencias con Acentuación en Microbiología) UANL, 2011.

Producción de tripsina de camarón blanco del pacífico (Litopenaeus vannamei) en Pichia pastoris

Tesis ( Doctorado en Ciencias con Especialidad en Biotecnología) UANL