Proteome analysis of Escherichia coli periplasmic proteins in response to over-expression of recombinant human interferon α2b

The periplasmic proteome of recombinant E. coli cells expressing human interferon-α2b (INF-α2b) was analysed by 2D-gel electrophoresis to find the most altered proteins. Of some unique up- and down-regulated proteins in the proteome, ten were identified by MS. The majority of the proteins belonged to the ABC transporter protein family. Other affected proteins were ones involved in the regulation of transcription such as DNA-binding response regulator, stress-related proteins and ecotin. Thus, the production of INF-α2b acts as a stress on the cells and results in the induction of various transporters and stress related proteins

Monoclonal anti-endoglin antibody TRC105 (carotuximab) prevents hypercholesterolemia and hyperglycemia-induced endothelial dysfunction in human aortic endothelial cells

15 p.-11 fig.Endoglin (Eng) is a co-receptor of the transforming growth factor β superfamily playing an important role in endothelial dysfunction. TRC105 (carotuximab) is a monoclonal antibody that blocks Eng and its downstream Smad signaling pathway. Here we have investigated for the first time the effects of TRC105 treatment on the development of endothelial dysfunction induced by 7-ketocholesterol (7K) or high glucose (HG), focusing on Eng expression, signaling, and function. In the hypercholesterolemia study, human aortic endothelial cells (HAoECs) were treated with TRC105 (300 μg/ml) for 1 h, followed by the addition of 7K (10 μg/ml) for another 12 h. In the hyperglycemia study, HAoECs were exposed to HG (45 mM) for 60 h, followed by the addition of TRC105 for another 12 h, and cells treated with 5mM glucose and 40 mM mannitol served as control. Protein levels, adhesion, and transmigration of monocytes were assessed by flow cytometry, mRNA expression was measured by qRT-PCR. 7K and HG treatment increased protein levels of NF-κB and Eng and adhesion and transmigration of monocytes through HAoECs monolayer. TRC105 pretreatment reduced the 7K- or HG-induced Eng protein levels and pSmad1/5 and pSmad2/3 signaling. Despite increased protein levels of P-selectin and VCAM-1, TRC105 mediated blockage of Eng prevented 7K- and HG-induced adhesion and transmigration of monocytes through endothelial monolayers. These results suggest that TRC105-mediated Eng blockage can counteract the hypercholesterolemia- and hyperglycemia-induced endothelial dysfunction in HAoECs, suggesting that Eng might be a potential therapeutic target in disorders associated with elevated cholesterol and glucose levels.This research was supported by grants from the Grant Agency of Charles University (GAUK No. 1130120), Czech Science Foundation (GACR 22-14961S), Specific University Research (SVV 260 549), efficiency and safety improvement of current drugs and nutraceuticals: advanced methods new challenge grant (EFSA-CDN; No. CZ.02.1.01/0.0/0.0/16_019/0000841), and by Consejo Superior de Investigaciones Científicas (CSIC), grant number: 201920E022.Peer reviewe

Expression of human epidermal growth factor in pichia pastoris and optimization of fermentation conditions

Human epidermal growth factor (hEGF) is a 6.2 kD polypeptide secreted by human tissues into the body fluid. It carries multiple biological functions including induction of tissue proliferation and differentiation, a process that is important in tissue repair. Because of this function, hEGF is widely used in various medical and industrial applications such as for wound healing, corneal transplant and treatment of gastric ulcers. As a result, the demand for hEGF supply for local consumption is always high, and there is a need for enough and cheap local supply of this protein. However, local production of hEGF especially in large commercial scales has not been successful due to various technical challenges. This study was carried out to express the hEGF gene in the yeast, Pichia pastoris GS115 using a commercially available expression vector equipped with a specific signal peptide that directs the transportation of the hEGF protein into the culture medium. The performance of the expression system for hEGF production was enhanced by optimizing the fermentation conditions of the yeast. The hEGF gene was ligated into the pPIC9K expression vector, and then integrated into the yeast’s genome by electroporation. The hEGF was assayed by using an ELISA-based system and the recombinant cell line that produced the highest hEGF was then used in the optimization of the fermentation conditions. In addition, the amount of methanol, an inducer for the expression system was also optimised. The highest amount of hEGF secreted into the medium was obtained from clone B1 of the recombinant yeast with an average yield of 2.27 μg/ml. This was obtained through an induction of the yeast culture with 0.5% (v/v) methanol for 60 hours. The best medium for the optimal hEGF production was BMMY buffered at a pH range of 6.0 and 7.0. Within this pH range, the difference in hEGF production was not significant. Two fermentation conditions commonly known to affect heterologous protein production (pH and temperature) were analyzed by using the artificial neural network (ANN). Changes in both pH and temperature significantly affected the hEGF production with the pH change had slightly higher impact on hEGF production than variations in the temperature. As anticipated from the model, the production of hEGF was decreased when the pH of the culture medium was lower than 6.0, or when the temperature was lower than 30°C. As a conclusion, hEGF was correctly secreted into the culture media of the recombinant P. pastoris as anticipated from the properties of the expression vector used. The amount of hEGF secreted into the medium was affected by the fermentation conditions especially the types of culture media used, the pH of the culture media and incubation temperature

High Soluble Endoglin Levels Affect Aortic Vascular Function during Mice Aging

Endoglin is a 180 kDa transmembrane glycoprotein that was demonstrated to be present in two different endoglin forms, namely membrane endoglin (Eng) and soluble endoglin (sEng). Increased sEng levels in the circulation have been detected in atherosclerosis, arterial hypertension, and type II diabetes mellitus. Moreover, sEng was shown to aggravate endothelial dysfunction when combined with a high-fat diet, suggesting it might be a risk factor for the development of endothelial dysfunction in combination with other risk factors. Therefore, this study hypothesized that high sEng levels exposure for 12 months combined with aging (an essential risk factor of atherosclerosis development) would aggravate vascular function in mouse aorta. Male transgenic mice with high levels of human sEng in plasma (Sol-Eng+) and their age-matched male transgenic littermates that do not develop high soluble endoglin (Control) on a chow diet were used. The aging process was initiated to contribute to endothelial dysfunction/atherosclerosis development, and it lasted 12 months. Wire myograph analysis showed impairment contractility in the Sol-Eng+ group when compared to the control group after KCl and PGF2α administration. Endothelium-dependent responsiveness to Ach was not significantly different between these groups. Western blot analysis revealed significantly decreased protein expression of Eng, p-eNOS, and ID1 expression in the Sol-Eng+ group compared to the control group suggesting reduced Eng signaling. In conclusion, we demonstrated for the first time that long-term exposure to high levels of sEng during aging results in alteration of vasoconstriction properties of the aorta, reduced eNOS phosphorylation, decreased Eng expression, and altered Eng signaling. These findings suggest that sEng can be considered a risk factor for the development of vascular dysfunction during aging and a potential therapeutical target for pharmacological intervention