scholarly journals Screening of CHO-K1 endogenous promoters for expressing recombinant proteins in mammalian cell cultures

2021 ◽  
Author(s):  
Ileana Tossolini ◽  
Agustina Gugliotta ◽  
Fernando Lopez Diaz ◽  
Ricardo Kratje ◽  
Claudio Prieto
Keyword(s):  
Recombinant Proteins ◽  
Mammalian Cells ◽  
High Strength ◽  
High Rate ◽  
High Expression ◽  
Expression Vectors ◽  
Cmv Promoter ◽  
Promising Alternative ◽  
Expression Levels ◽  
Synthetic Promoters

For the production of recombinant protein therapeutics in mammalian cells, a high rate of gene expression is desired and hence strong viral-derived promoters are commonly used. However, they usually induce cellular stress and can be susceptible to epigenetic silencing. Endogenous promoters, which coordinates their activity with cellular and bioprocess dynamics while at the same time they maintain high expression levels, may help to avoid such drawbacks. In this work, endogenous promoters were identified based on high expression levels in RNA-seq data of CHO-K1 cells cultured in high density. The promoters of Actb, Ctsz, Hmox1, Hspa5, Vim and Rps18 genes were selected for generating new expression vectors for the production of recombinant proteins in mammalian cells. The in silico derived promoter regions were experimentally verified and the majority showed transcriptional activity comparable or higher than CMV. Also, stable expression following a reduction of culture temperature was investigated. The characterized endogenous promoters (excluding Rps18) constitute a promising alternative to CMV promoter due to their high strength, long-term expression stability and integration into the regulatory network of the host cell. These promoters may also comprise an initial panel for designing cell engineering strategies and synthetic promoters, as well as for industrial cell line development.

scholarly journals The “beauty in the beast”—the multiple uses of Priestia megaterium in biotechnology

2021 ◽  
Author(s):  
Rebekka Biedendieck ◽  
Tobias Knuuti ◽  
Simon J. Moore ◽  
Dieter Jahn
Keyword(s):  
Small Molecules ◽  
Recombinant Proteins ◽  
Value Added ◽  
Expression Vectors ◽  
Translation System ◽  
Vitamin B ◽  
Cell Factory ◽  
Promising Alternative ◽  
In Vitro Synthesis ◽  
Whole Cell

Abstract Over 30 years, the Gram-positive bacterium Priestia megaterium (previously known as Bacillus megaterium) was systematically developed for biotechnological applications ranging from the production of small molecules like vitamin B12, over polymers like polyhydroxybutyrate (PHB) up to the in vivo and in vitro synthesis of multiple proteins and finally whole-cell applications. Here we describe the use of the natural vitamin B12 (cobalamin) producer P. megaterium for the elucidation of the biosynthetic pathway and the subsequent systematic knowledge-based development for production purposes. The formation of PHB, a natural product of P. megaterium and potential petro-plastic substitute, is covered and discussed. Further important biotechnological characteristics of P. megaterium for recombinant protein production including high protein secretion capacity and simple cultivation on value-added carbon sources are outlined. This includes the advanced system with almost 30 commercially available expression vectors for the intracellular and extracellular production of recombinant proteins at the g/L scale. We also revealed a novel P. megaterium transcription-translation system as a complementary and versatile biotechnological tool kit. As an impressive biotechnology application, the formation of various cytochrome P450 is also critically highlighted. Finally, whole cellular applications in plant protection are completing the overall picture of P. megaterium as a versatile giant cell factory. Key points • The use of Priestia megaterium for the biosynthesis of small molecules and recombinant proteins through to whole-cell applications is reviewed. • P. megaterium can act as a promising alternative host in biotechnological production processes.

scholarly journals Model-guided quantitative analysis of microRNA-mediated regulation on competing endogenous RNAs using a synthetic gene circuit

2015 ◽  
Vol 112 (10) ◽  
pp. 3158-3163 ◽  
Author(s):  
Ye Yuan ◽  
Bing Liu ◽  
Peng Xie ◽  
Michael Q. Zhang ◽  
Yanda Li ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
High Expression ◽  
Synthetic Gene ◽  
Expression Levels ◽  
Synthetic Gene Circuits ◽  
Sirna Design ◽  
Competing Endogenous Rnas ◽  
Cultured Human Cells ◽  
Quantitative Understanding ◽  
Target Effect

Competing endogenous RNAs (ceRNAs) cross-regulate each other at the posttranscriptional level by titrating shared microRNAs (miRNAs). Here, we established a computational model to quantitatively describe a minimum ceRNA network and experimentally validated our model predictions in cultured human cells by using synthetic gene circuits. We demonstrated that the range and strength of ceRNA regulation are largely determined by the relative abundance and the binding strength of miRNA and ceRNAs. We found that a nonreciprocal competing effect between partially and perfectly complementary targets is mainly due to different miRNA loss rates in these two types of regulations. Furthermore, we showed that miRNA-like off targets with high expression levels and strong binding sites significantly diminish the RNA interference efficiency, but the effect caused by high expression levels could be compensated by introducing more small interference RNAs (siRNAs). Thus, our results provided a quantitative understanding of ceRNA cross-regulation via shared miRNA and implied an siRNA design strategy to reduce the siRNA off-target effect in mammalian cells.

scholarly journals Artificial introns for effective expression of transgenes in mammalian cells

2021 ◽  
Author(s):  
D.S. Naberezhnov ◽  
E.A. Lesovaya ◽  
K.I. Kirsanov ◽  
M.G. Yakubovskaya
Keyword(s):  
Gene Expression ◽  
Recombinant Proteins ◽  
Mammalian Cells ◽  
Site Directed Mutagenesis ◽  
High Expression ◽  
Expression Level ◽  
Eukaryotic Cells ◽  
Directed Mutagenesis ◽  
Large Size ◽  
High Expression Level

AbstractIntrons are widely used in the assembly of genetic constructions expressing transgenic proteins in eukaryotic cells for the enhancement of this expression. However, the choice of introns that can be applied for such purposes is limited by the excessively large size of the majority of natural introns (several thousand nucleotides) and therefore they cannot be cloned in a genetic construction. With the help of site-directed mutagenesis we have generated a library of short (99 nucleotides long) introns. The efficiency of these introns in the enhancement of gene expression was analyzed. As a result, a set of 12 introns was selected. The generated intros can be used for genetic constructions with high expression level of recombinant proteins.

scholarly journals Control of Multigene Expression Stoichiometry in Mammalian Cells Using Synthetic Promoters

2021 ◽  
Author(s):  
Yash D. Patel ◽  
Adam J. Brown ◽  
Jie Zhu ◽  
Guglielmo Rosignoli ◽  
Suzanne J. Gibson ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Synthetic Promoters ◽  
Multigene Expression

scholarly journals Utilization of Polymer Concrete Composites for a Circular Economy: A Comparative Review for Assessment of Recycling and Waste Utilization

Polymers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 2135
Author(s):  
Hatem Alhazmi ◽  
Syyed Adnan Raheel Shah ◽  
Muhammad Kashif Anwar ◽  
Ali Raza ◽  
Muhammad Kaleem Ullah ◽  
...  
Keyword(s):  
Polymer Composites ◽  
Polymer Composite ◽  
Circular Economy ◽  
Agricultural Waste ◽  
High Strength ◽  
Supplementary Cementitious Materials ◽  
Polymer Concrete ◽  
Promising Alternative ◽  
Detailed Review ◽  
Comparative Review

Polymer composites have been identified as the most innovative and selective materials known in the 21st century. Presently, polymer concrete composites (PCC) made from industrial or agricultural waste are becoming more popular as the demand for high-strength concrete for various applications is increasing. Polymer concrete composites not only provide high strength properties but also provide specific characteristics, such as high durability, decreased drying shrinkage, reduced permeability, and chemical or heat resistance. This paper provides a detailed review of the utilization of polymer composites in the construction industry based on the circular economy model. This paper provides an updated and detailed report on the effects of polymer composites in concrete as supplementary cementitious materials and a comprehensive analysis of the existing literature on their utilization and the production of polymer composites. A detailed review of a variety of polymers, their qualities, performance, and classification, and various polymer composite production methods is given to select the best polymer composite materials for specific applications. PCCs have become a promising alternative for the reuse of waste materials due to their exceptional performance. Based on the findings of the studies evaluated, it can be concluded that more research is needed to provide a foundation for a regulatory structure for the acceptance of polymer composites.

scholarly journals Baicalin Represses Type Three Secretion System of Pseudomonas aeruginosa through PQS System

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1497
Author(s):  
Pansong Zhang ◽  
Qiao Guo ◽  
Zhihua Wei ◽  
Qin Yang ◽  
Zisheng Guo ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Virulence Factors ◽  
Mammalian Cells ◽  
Secretion System ◽  
Chinese Herbs ◽  
Infection Model ◽  
Lung Pathology ◽  
Promising Alternative ◽  
The Impact

Therapeutics that target the virulence of pathogens rather than their viability offer a promising alternative for treating infectious diseases and circumventing antibiotic resistance. In this study, we searched for anti-virulence compounds against Pseudomonas aeruginosa from Chinese herbs and investigated baicalin from Scutellariae radix as such an active anti-virulence compound. The effect of baicalin on a range of important virulence factors in P. aeruginosa was assessed using luxCDABE-based reporters and by phenotypical assays. The molecular mechanism of the virulence inhibition by baicalin was investigated using genetic approaches. The impact of baicalin on P. aeruginosa pathogenicity was evaluated by both in vitro assays and in vivo animal models. The results show that baicalin diminished a plenty of important virulence factors in P. aeruginosa, including the Type III secretion system (T3SS). Baicalin treatment reduced the cellular toxicity of P. aeruginosa on the mammalian cells and attenuated in vivo pathogenicity in a Drosophila melanogaster infection model. In a rat pulmonary infection model, baicalin significantly reduced the severity of lung pathology and accelerated lung bacterial clearance. The PqsR of the Pseudomonas quinolone signal (PQS) system was found to be required for baicalin’s impact on T3SS. These findings indicate that baicalin is a promising therapeutic candidate for treating P. aeruginosa infections.

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