title: An EPR study of the secretion of G-CSF heterologous protein from Pichia pastoris.
abstract: The biologically active protein known as human granulocyte colony stimulating factor (G-CSF) can be efficiently secreted from the transformed GS115 Pichia pastoris (GS115/pPIC9/G-CSF), which contains an alpha-mating-factor prepro signal sequence and an alcohol oxidase I promoter, both introduced using the pPIC9 plasmid. To study the P. pastoris G-CSF protein-secretion process, changes to the plasma membrane's lateral domain structure were monitored using electron paramagnetic resonance (EPR). The GS115 and its transformed analogue show that the plasma membrane can be described by fluid-disordered and fluid-ordered lateral domains. The relative proportion of these domains is a sensitive parameter that is able to describe the membrane's involvement in the protein-excretion process. Here, P. pastoris GS115 served as a control for us to compare with the GS115/pPIC9/G-CSF heterologous protein-secreting cells. Electron paramagnetic resonance studies using the spin-probe 5-doxyl methyl ester of palmitic acid [MeFASL (10,3)] showed that during cultivation in a glycerol medium all types of cells had a relatively high proportion of cell-membrane fluid-disordered domains, while during the production phase the G-CSF protein-secreting cells showed a decrease in the proportion of fluid-disordered domains. We ascribe this effect to the vesicular lipid exchange caused by the fusion of secretary vesicles with the plasma membrane during exocytosis. Using electron microscopy and immunocytochemistry intracellular vesicles containing the G-CSF protein were detected. Our studies support the exocytotic mechanism of the heterologous protein secretion.
authors.affiliation: Lek,d.d., R&D, Department of Biotechnology, Ljubljana, Slovenia.
authors.forename: M
authors.initials: M
authors.lastname: Anzur Lasnik
authors.forename: M
authors.initials: M
authors.lastname: Nemec
authors.forename: R
authors.initials: R
authors.lastname: Romih
authors.forename: M
authors.initials: M
authors.lastname: Schara
mesh_terms: D002463:Cell Membrane Permeability / Q000502:physiology; D019976:Cloning, Organism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D016179:Granulocyte Colony-Stimulating Factor / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008560:Membrane Fluidity / Q000502:physiology; D021962:Membrane Microdomains / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D010843:Pichia / Q000737:chemistry / Q000254:growth & development / Q000378:metabolism* / Q000648:ultrastructure; D011994:Recombinant Proteins
chemical_list: D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor