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

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.

HSF-1, HIF-1and HSP90 expression on recombinant Pichia pastoris under fed-batch fermentation

Pichia pastoris is a methylotrophic yeast used as an efficient expression system for heterologous protein production as compared to other expression systems. Considering that every cell must respond to environmental changes to survive and differentiate, determination of endogenous protein related to heat stress responses and hypoxia, it would necessary to establish the temperature and methanol concentration conditions for optimal growth. The aim of this study is characterize the culture conditions through the putative biomarkers in different conditions of temperature and methanol concentration. 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. The western blot results of HIF-1α and HSP-90 did not indicate statistically significant in the culture conditions studied. Respect to biomarkers location, HIF-1α and HSP-90 presented differences between cultures. In conclusion, the results suggest the cultures in a hypoxic condition produce a high density and yeast cells smaller. Beside the high density would not necessary related with a high production of recombinant proteins in modified-genetically P. pastoris.

Expression of Heat Stress Protein 70 mRNA in Patients with Chronic Ob-structive Pulmonary Disease and Its Significance / 华中科技大学学报（医学）（英德文版）

The effects of cigarette smoke extract (CSE) on the expression of heat stress protein 70(Hsp70) in human bronchi smooth muscle cells were investigated in vitro, and the changes in Hsp70 mRNA in the patients with chronic obstructive pulmonary disease and their significance were explored. Human bronchi smooth muscle cells were cultured with CSE at the different concentrations. The expression of Hsp70 mRNA and Hsp70 was detected by reverse translation-polymerase chain reaction (RT-PCR) and Western blotting respectively. Levels of Hsp70 mRNA and Hsp70 in lymphocytes from 20 patients with COPD and 20 healthy smoking control subjects were measured by RT-PCR and Western blotting. The results showed the expression of both Hsp70 mRNA and Hsp70 was decreased conformably in human bronchi smooth muscle cells treated with CSE at certain concentration in vitro. The A values of the Hsp70 mRNA expression were 0. 24±0.11 and 0.42±0.13 respectively in COPD patients and healthy smoking controls with the difference being significant (P＜0.01). There was also significant difference in the A values of the Hsp70 expression between COPD patients and healthy smoking controls (20.9±9.9 vs 44.8±15.3, P＜0.01). The levels of Hsp70 mRNA had strongly positive correlation with Hsp70 protein (r = 0. 85, P＜0.01). It was suggested that the expression of Hsp70 mRNA was in concordance with the expression of Hsp70, which could provide a basis on the study of Hsp70 gene regulation and Hsp70 gene in the development of COPD.

The Effect of Dehydration after a Sauna and Intermittent Hypoxia Stimuli on Heat Stress Protein 70 in Human Leucocytes and Aerobic Capacity during Incremental Exercise / 中国运动医学杂志

The purpose of this study was to compare Heat Stress Protein70 (HSP70) in human leucocytes, VO2max and Blood Lactate (BLa) during incremental exercise under a thermoneutral condition without taking a sauna with those during the same workload and under same thermoneutral condition after a sauna and after intermittent hypoxia stimuli. Ten unacclimated men performed an incremental test to exhaustion on a treadmill under a thermoneutral condition without taking a sauna (N25℃, relative humidity 65%) and during the same workload and under same thermoneutral condition after a sauna (D25℃, relative humidity 65%), and after intermittent hypoxia stimuli (LN25℃, relative humidity 65%) . The results were as follows: (1) HSP70 levels before incremental exercise in D25℃ were significantly higher than that in N25℃(13701.87?5367.17vs 7517.57?1980.01,P