Biomarkers to evaluate the effects of temperature and methanol on recombinant Pichia pastoris

Autor(es)

Zepeda, Andrea B. ^{[1, 2]} ; Figueroa, Carolina A. ^{[1, 2]} ; Abdalla, Dulcineia S. P. ^[3] ; Maranhao, Andrea Q. ^[4] ; Ulloa, Patricio H. ^[2] ; Pessoa, Jr., Adalberto ^[1] ; Farias, Jorge G. ^[2]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Fac Ciencias Farmaceut, Dept Tecnol Bioquim Farmaceut, Sao Paulo - Brazil

^[2] Univ La Frontera, Fac Ingn Ciencias & Adm, Dept Ingn Quim, Temuco - Chile

^[3] Univ Sao Paulo, Fac Ciencias Farmaceut, Dept Anal Clin & Toxicol, Sao Paulo - Brazil

^[4] Univ Brasilia, Inst Ciencias Biol, Dept Biol Celular, Brasilia, DF - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Brazilian Journal of Microbiology; v. 45, n. 2, p. 475-483, APR-JUN 2014.
• Citações Web of Science: 4

Resumo

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 degrees 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 degrees C, 4X = 3% methanol-30 degrees C, and 5X = 1% methanol- 10 degrees C. The expression level of HIF-1 alpha, 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 degrees 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 degrees C and 1% and at methanol -10 degrees 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 degrees C is a condition that induces a strong oxidative stress and risk factors of apoptosis in modified-genetically P. pastoris. (AU)

• Processo FAPESP: 12/50210-9 - Produção de fragmentos recombinantes de anticorpos a partir do cultivo de Pichia pastoris geneticamente modificada
• Beneficiário: Adalberto Pessoa Junior
• Modalidade de apoio: Auxílio à Pesquisa - Regular