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1.
The EF‐1α promoter maintains high‐level transgene expression from episomal vectors in transfected CHO‐K1 cells 下载免费PDF全文
Xi Zhang Danhua Xu Qin Li Junhe Zhang Tianyun Wang 《Journal of cellular and molecular medicine》2017,21(11):3044-3054
In our previous study, we demonstrated that episomal vectors based on the characteristic sequence of matrix attachment regions (MARs) and containing the cytomegalovirus (CMV) promoter allow transgenes to be maintained episomally in Chinese hamster ovary (CHO) cells. However, the transgene expression was unstable and the number of copies was low. In this study, we focused on enhancers, various promoters and promoter variants that could improve the transgene expression stability, expression magnitude (level) and the copy number of a MAR‐based episomal vector in CHO‐K1 cells. In comparison with the CMV promoter, the eukaryotic translation elongation factor 1 α (EF‐1α, gene symbol EEF1A1) promoter increased the transfection efficiency, the transgene expression, the proportion of expression‐positive clones and the copy number of the episomal vector in long‐term culture. By contrast, no significant positive effects were observed with an enhancer, CMV promoter variants or CAG promoter in the episomal vector in long‐term culture. Moreover, the high‐expression clones harbouring the EF‐1α promoter tended to be more stable in long‐term culture, even in the absence of selection pressure. According to these findings, we concluded that the EF‐1α promoter is a potent regulatory sequence for episomal vectors because it maintains high transgene expression, transgene stability and copy number. These results provide valuable information on improvement of transgene stability and the copy number of episomal vectors. 相似文献
2.
Signal peptides are short peptides located at the N-terminus of secreted proteins. They characteristically have three domains; a basic region at the N-terminus (n-region), a central hydrophobic core (h-region) and a carboxy-terminal cleavage region (c-region). Although hundreds of different signal peptides have been identified, it has not been completely understood how their features enable signal peptides to influence protein expression. Antibody-derived signal peptides are often used to prepare recombinant antibodies expressed by eukaryotic cells, especially Chinese hamster ovary (CHO) cells. However, when prokaryotic Escherichia coli (E. coli) are utilized in drug discovery processes, such as for phage display selection or antibody humanization, signal peptides have been selected separately due to the differences in the expression systems between the species. In this study, we successfully established a signal peptide that enables a functional antibody to be expressed in both prokaryotic and eukaryotic cells by focusing on the importance of having an Ala residue in the c-region of the signal sequence. We found that changing Ser to Ala at only two positions significantly augmented the anti-HER2 antigen binding fragment (Fab) expression in E. coli. In addition, this altered signal peptide also retained the ability to express functional anti-HER2 antibody in CHO cells. Taken together, the present findings indicate that the signal peptide can promote functional antibody expression in both prokaryotic E. coli and eukaryotic CHO cells. This finding will contribute to the understanding of signal peptides and accelerate therapeutic antibody research. 相似文献
3.
Li Liu Jingang Cui Qinbo Yang Chenglin Jia Minqi Xiong Bingbing Ning Xiaoye Du Peiwei Wang Xintong Yu Li Li Wenjian Wang Yu Chen Teng Zhang 《Biochemical and biophysical research communications》2014
Oxidative stress is mechanistically implicated in the pathogenesis of myocardial injury and the subsequent fibrogenic tissue remodeling. Therapies targeting oxidative stress in the process of myocardial fibrogenesis are still lacking and thus remain as an active research area in myocardial injury management. The current study evaluated the effects of a NADPH oxidase inhibitor, apocynin, on the production of reactive oxygen species and the development of myocardial fibrogenesis in isoproterenol (ISO)-induced myocardial injury mouse model. The results revealed a remarkable effect of apocynin on attenuating the development of myocardial necrotic lesions, inflammation and fibrogenesis. Additionally, the protective effects of apocynin against myocardial injuries were associated with suppressed expression of an array of genes implicated in inflammatory and fibrogenic responses. Our study thus provided for the first time the histopathological and molecular evidence supporting the therapeutic value of apocynin against the development of myocardial injuries, in particular, myocardial fibrogenesis, which will benefit the mechanism-based drug development targeting oxidative stress in preventing and/or treating related myocardial disorders. 相似文献
4.
Georgios Tzelepis Akira Hosomi Tanim Jabid Hossain Hiroto Hirayama Mukesh Dubey Dan Funck Jensen Tadashi Suzuki Magnus Karlsson 《Biochemical and biophysical research communications》2014
N-Glycosylation is an important post-translational modification of proteins, which mainly occurs in the endoplasmic reticulum (ER). Glycoproteins that are unable to fold properly are exported to the cytosol for degradation by a cellular system called ER-associated degradation (ERAD). Once misfolded glycoproteins are exported to the cytosol, they are subjected to deglycosylation by peptide:N-glycanase (PNGase) to facilitate the efficient degradation of misfolded proteins by the proteasome. Interestingly, the ortholog of PNGase in some filamentous fungi was found to be an inactive deglycosylating enzyme. On the other hand, it has been shown that in filamentous fungi genomes, usually two different fungi-specific endo-β-N-acetylglucosamidases (ENGases) can be found; one is predicted to be localized in the cytosol and the other to have a signal sequence, while the functional importance of these enzymes remains to be clarified. In this study the ENGases of the filamentous fungus Trichoderma atroviride was characterized. By heterologous expression of the ENGases Eng18A and Eng18B in Saccharomyces cerevisiae, it was found that both ENGases are active deglycosylating enzymes. Interestingly, only Eng18B was able to enhance the efficient degradation of the RTL protein, a PNGase-dependent ERAD substrate, implying the involvement of this enzyme in the ERAD process. These results indicate that T. atroviride Eng18B may deglycosylate misfolded glycoproteins, substituting the function of the cytoplasmic PNGase in the ERAD process. 相似文献
5.
The heterologous recombinant expression of proteins in Escherichia coli without start–methionine is a common problem. The nitrophorin 7 heme properties and function strongly depend on the accurate N-terminal amino acid sequence. Leading protein expression into the periplasm by fusion with the leader peptide pelB yields functional protein; however, the folded protein sticks to the cell debris. Therefore, the periplasmic fraction was dissolved in guanidinium chloride and folded by a drop-in method. Separation from impurities including residual pelB–nitrophorin 7 required establishing an unconventional chromatographic technique using calcium-loaded Chelating Sepharose as cation exchanger and elution by a linear CaCl2 gradient. 相似文献
6.
Promoter analysis typically employs a reporter gene fused to a test promoter combined with a second reporter fused to a control promoter that is used for normalization purposes. However, this approach is not valid when experimental conditions affect the control promoter. We have developed and validated a single secreted luciferase reporter (SSLR) assay for promoter analysis that avoids the use of a control reporter. The approach uses an early level of expression of a secreted luciferase linked to a test promoter as an internal normalization control for subsequent analysis of the same promoter. Comparison of the SSLR assay with the dual luciferase reporter (DLR) assay using HMGCR (3-hydroxy-3-methylglutaryl-coenzyme A reductase) and LDLR (low-density lipoprotein receptor) promoter constructs, which are down-regulated by 25-hydroxycholesterol, show that both assays yield similar results. Comparison of the response of the HMGCR promoter in SSLR transient assays compared very favorably with the response of the same promoter in the stable cell line. Overall, the SSLR assay proved to be a valid alternative to the DLR assay for certain applications and had significant advantages in that measurement of only one luciferase is required and monitoring can be continuous because cell lysis is not necessary. 相似文献
7.
Andrew G. Cridge Jyothsna Visweswaraiah Rashmi Ramesh Evelyn Sattlegger 《Analytical biochemistry》2014
This work describes a quick semi-quantitative colony immunoassay (QSCI) method for immunoblot detection of intracellularly expressed proteins in both yeast and bacterial cells. After induction of protein expression, only 4.5 h is required for cell breakage, protein detection, and data analysis. This protocol was used to screen and unambiguously identify Saccharomyces cerevisiae cells efficiently overexpressing glutathione S-transferase (GST)-tagged Yih1 in addition to cells expressing the myc-tagged large 297-kDa Gcn1 protein. In addition, the method was used to identify Escherichia coli cells efficiently expressing His6-tagged Yih1 and a GST-tagged Gcn1 fragment, respectively. The protocol allows the use of both epitope-specific and protein-specific antibodies. The same colony immunoassay can also be used to determine the minimal concentration of inducing agent sufficient for induction of optimal protein expression (e.g., galactose for yeast, isopropyl β-d-1-thiogalactopyranoside [IPTG] for E. coli). To our knowledge, this is the first report on a rapid low-cost procedure that allows the calibration of inducing agent on solid medium. 相似文献
8.
运用相邻格子法及聚集度指标测定法,在植被调查的基础上,对云南普洱不同恢复阶段(恢复15a、30a和老龄林)季风常绿阔叶林的刺栲(Castanopsis hystrix)、短刺栲(Castanopsis echidnocarpa)和红木荷(Schima wallichii)3个优势物种进行种群空间分布格局研究,以阐明种群分布格局与生境条件、种群年龄结构的动态关系,揭示种群发育过程中空间动态与生物学机理。结果发现:在所有恢复阶段内,刺栲、短刺栲、红木荷均为聚集分布。在不同尺度下,刺栲在原始林中10m×10m尺度下为均匀分布,而在恢复15年中20m×20m尺度为随机分布;红木荷在10m×20m尺度下为均匀分布,在其它所有恢复阶段和尺度下3个物种均为聚集分布。研究结果表明,不同恢复阶段和不同尺度下刺栲、短刺栲和红木荷3个优势物种绝大多数为聚集分布,说明聚集分布为3个优势种的基本属性。 相似文献
9.
选取我国东北、西南、西北及中北部地区的10个典型湖泊,调查了表层沉积物生物硅含量变化情况,并通过区域对比分析了生物硅含量变化的原因,寻找我国湖泊生物硅变化的空间规律。结果表明,西北及中北部地区湖泊生物硅含量平均值整体低于东北和西南地区湖泊,可能是由于西北及中北部地区较低的温度和较少的降水量导致硅藻生长受限,从而造成生物硅含量偏低。东北湖泊与西南湖泊生物硅含量相对较高,但其形成原因不同。东北2个湖泊均靠近人类聚居区,属富营养型湖泊,虽然东北地区年均温较低,但充足的营养盐为硅藻生长提供了必要因素。而西南地区湖泊大多为中贫营养型湖泊,其生物硅含量整体较高可能主要归因于较高的温度和较多的降水。 相似文献