Construction of a dietary-cure Saccharomyces cerevisiae expressing long-acting glucagon-like peptide-1 and investigation of its hypoglycemic activity in type 2 diabetes mellitus mouse model

Bio-drug is a new type of beneficial biology expressing therapeutic peptides (protein) as orally administrated medicine totreat diseases, in particular, chronic diseases like diabetes. In order to develop recombinant yeast strains as a bio-drug whichcould effectively ameliorate type 2 diabetes, an integrating expression vector pNK1-PGK that could successfully expressgreen fluorescent protein (GFP) in Saccharomyces cerevisiae was constructed to demonstrate the normality of the function.A pNK1-PGK vector, which expresses 10 tandem repeats of long-acting glucagon-like peptide-1(10laGLP-1), was clonedand then transformed into the S. cerevisiae INVSc1. The long-acting GLP-1 hypoglycemic yeast (LHY168) that grewrapidly and expressed 10laGLP-1 stably was screened by uracil-deficient plates and Western blot. The expression quantityof 10laGLP-1 reached 1.56 mg/g cell wet weight. Moreover, the oral administration of LHY168 significantly declined theblood glucose in type 2 diabetic mice that were constructed through co-induction of streptozotocin (STZ) and high-fat andhigh-sugar diet.

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.

Application of Gene Recombinant Yeast Cell in Screening to Environmental Estrogen / 环境与健康杂志

Objective To research the estrogen-like activity of the DDTs and the extract of vehicle exhaust by recombinant yeast system.Methods Being added into the yeast cell culture fluid,the objects reached the final concentration of 10-9～10-15 mol/L for 17?-estradiol,10-4～10-9 mol/L for DDTs(p,p'-DDT,o,p'-DDT,p,p'-DDD,p,p'-DDE),0.25～8.00 ml/ml for the extract of vehicle exhaust,then the cultivation was performed for 4 hours,the quantitative assay of ?-galactosidase activity accounts for the estrogenlike effect of the samples was carried out.Results DDTs were a group of environmental estrogens hormones,which could integrate estrogen receptors as the stimulators do and express the estrogen-like effect.Being tested separately,the DDTs showed the estrogen-like activity,which was stronger for o,p'-DDT and weaker for p,p'-DDE.The?-galactosidase activity of gasoline-fueled vehicle exhaust at the final concentration of 2.00～4.00 ml/ml was significantly different from that of the DMSO(P

Additive Estrogenic Activities of the Binary Mixtures of Four Estrogenic Chemicals in Recombinant Yeast Expressing Human Estrogen Receptor

To evaluate the estrogenic activities of several chemicals such as 17 beta-estradiol (E2), rho-nonylphenol, bisphenol A, butylparaben, and combinations of these chemicals, we used recombinant yeasts containing the human estrogen receptor [Saccharomyces cerevisiae ER + LYS 8127]. We evaluated E2 was most active in the recombinant yeast assay, followed by rho-nonylphenol, bisphenol A, butylparaben. The combinations of some concentrations of 17-estradiol as a strong estrogen and bisphenol A or butylparaben as a weak estrogen showed additive estrogenic effects. Also, the combinations of some concentrations of nonlyphenol and butylparaben and combination of butylparaben and bisphenol A showed additive effects in the estrogenic activity. Therefore, the estrogenic activities of the combinations of two chemicals were additive, not synergistic.