title: Biomarkers to evaluate the effects of temperature and methanol on recombinant Pichia pastoris.
abstract: Pichia pastoris is methylotrophic yeast used as an efficient expression system for heterologous protein production. In order to evaluate the effects of temperature (10 and 30 °C) and methanol (1 and 3% (v/v)) on genetically-modified Pichia pastoris, different biomarkers were evaluated: Heat stress (HSF-1 and Hsp70), oxidative stress (OGG1 and TBARS) and antioxidant (GLR). Three yeast cultures were performed: 3X = 3% methanol-10 °C, 4X = 3% methanol-30 °C, and 5X = 1% methanol-10°C. The expression level of HIF-1α, HSF-1, HSP-70 and HSP-90 biomarkers were measured by Western blot and in situ detection was performed by immunocytochemistry. Ours results show that at 3% methanol -30 °C there is an increase of mitochondrial OGG1 (mtOGG1), Glutathione Reductase (GLR) and TBARS. In addition, there was a cytosolic expression of HSF-1 and HSP-70, which indicates a deprotection against nucleolar fragmentation (apoptosis). On the other hand, at 3% methanol -10 °C and 1% and at methanol -10 °C conditions there was nuclear expression of OGG1, lower levels of TBARS and lower expression of GLR, cytosolic expression of HSF-1 and nuclear expression HSP-70. In conclusion, our results suggest that 3% methanol-30 °C is a condition that induces a strong oxidative stress and risk factors of apoptosis in modified-genetically P. pastoris.
authors.affiliation: Departamento de Ingeniería Química Facultad de Ingeniería, Ciencias y Administración Universidad de La Frontera Temuco Chile Departamento de Ingeniería Química, Facultad de Ingeniería, Ciencias y Administración, Universidad de La Frontera, Temuco, Chile. ; Departamento de Tecnologia Bioquímico-Farmacêutica Faculdade de Ciências Farmacêuticas Universidade de São Paulo São PauloSP Brazil Departamento de Tecnologia Bioquímico-Farmacêutica, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP, Brazil.
authors.forename: Andrea B
authors.initials: AB
authors.lastname: Zepeda
authors.affiliation: Departamento de Ingeniería Química Facultad de Ingeniería, Ciencias y Administración Universidad de La Frontera Temuco Chile Departamento de Ingeniería Química, Facultad de Ingeniería, Ciencias y Administración, Universidad de La Frontera, Temuco, Chile. ; Departamento de Tecnologia Bioquímico-Farmacêutica Faculdade de Ciências Farmacêuticas Universidade de São Paulo São PauloSP Brazil Departamento de Tecnologia Bioquímico-Farmacêutica, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP, Brazil.
authors.forename: Carolina A
authors.initials: CA
authors.lastname: Figueroa
authors.affiliation: Departamento de Análises Clínicas e Toxicológicas Faculdade de Ciências Farmacêuticas Universidade de São Paulo São PauloSP Brazil Departamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP, Brazil.
authors.forename: Dulcineia S P
authors.initials: DS
authors.lastname: Abdalla
authors.affiliation: Departamento de Biología Celular Instituto de Ciências Biológicas Universidade de Brasilia BrasíliaDF Brazil Departamento de Biología Celular, Instituto de Ciências Biológicas, Universidade de Brasilia, Brasília, DF, Brazil.
authors.forename: Andrea Q
authors.initials: AQ
authors.lastname: Maranhão
authors.affiliation: Departamento de Ingeniería Química Facultad de Ingeniería, Ciencias y Administración Universidad de La Frontera Temuco Chile Departamento de Ingeniería Química, Facultad de Ingeniería, Ciencias y Administración, Universidad de La Frontera, Temuco, Chile.
authors.forename: Patricio H
authors.initials: PH
authors.lastname: Ulloa
authors.affiliation: Departamento de Tecnologia Bioquímico-Farmacêutica Faculdade de Ciências Farmacêuticas Universidade de São Paulo São PauloSP Brazil Departamento de Tecnologia Bioquímico-Farmacêutica, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP, Brazil.
authors.forename: Adalberto
authors.initials: A
authors.lastname: Pessoa
authors.affiliation: Departamento de Ingeniería Química Facultad de Ingeniería, Ciencias y Administración Universidad de La Frontera Temuco Chile Departamento de Ingeniería Química, Facultad de Ingeniería, Ciencias y Administración, Universidad de La Frontera, Temuco, Chile.
authors.forename: Jorge G
authors.initials: JG
authors.lastname: Farías
mesh_terms: D000975:Antioxidants / Q000032:analysis; D015415:Biomarkers / Q000032:analysis*; D005656:Fungal Proteins / Q000032:analysis; D020869:Gene Expression Profiling; D006358:Hot Temperature; D000432:Methanol / Q000378:metabolism*; D018384:Oxidative Stress; D010843:Pichia / Q000187:drug effects* / Q000502:physiology / Q000528:radiation effects*; D013312:Stress, Physiological; D013696:Temperature
chemical_list: D000975:Antioxidants; D015415:Biomarkers; D005656:Fungal Proteins; D000432:Methanol
keywords: DNA damage; heat stress protein; oxidative stress; recombinant yeast