RESUMEN
Chinese hamster ovary (CHO) cells are currently the most widely used host cells for recombinant therapeutic protein (RTP) production. Currently, the RTP yields need to increase further to meet the market needs and reduce costs. In this study, three stabilizing and anti-repressor (SAR) elements from the human genome were selected, including human SAR7, SAR40, and SAR44 elements. SAR elements were cloned upstream of the promoter in the eukaryotic vector, followed by transfection into CHO cells, and were screened under G418 pressure. Flow cytometry was used to detect enhanced green fluorescent protein (eGFP) expression levels. The gene copy numbers and mRNA expression levels were determined through quantitative real-time PCR. Furthermore, the effect of the stronger SAR elements on adalimumab was investigated. The results showed that transgene expression levels in the SAR-containing vectors were higher than that of the control vector, and SAR7 and SAR40 significantly increased and maintained the long-term expression of the transgene in CHO cells. In addition, the transgene expression level increase was related with gene copy numbers and mRNA expression levels. Collectively, SAR elements can enhance the transgene expression and maintain the long-term expression of a transgene in transfected CHO cells, which may be used to increase recombinant protein production in CHO cells.
RESUMEN
Mammalian cells are the preferred choice system for the production of complex molecules, such as recombinant therapeutic proteins. Although the technology for increasing the yield of proteins has improved rapidly, the process of selecting, identifying as well as maintaining high-yield cell clones is still troublesome, time-consuming and usually uncertain. Optimization of expression vectors is one of the most effective methods for enhancing protein expression levels. Several commonly used chromatin-modifying elements, including the matrix attachment region, ubiquitous chromatin opening elements, insulators, stabilizing anti-repressor elements can be used to increase the expression level and stability of recombinant proteins. In this review, these chromatin-modifying elements used for the expression vector optimization in mammalian cells are summarized, and future strategies for the utilization of expression cassettes are also discussed.
Asunto(s)
Cromatina/genética , Ingeniería de Proteínas/métodos , Proteínas Recombinantes , Secuencias Reguladoras de Ácidos Nucleicos/genética , Animales , Células CHO , Línea Celular , Cricetinae , Cricetulus , Vectores Genéticos/genética , Células HEK293 , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Secretion of effector proteins in Enteropathogeneic Escherichia coli (EPEC) and Enterohemorrhagic Escherichia coli (EHEC) is mediated by a specialized type III secretion system, components of which are encoded in the LEE operons 1 to 5. H-NS, a global repressor in E. coli, silences the expression of LEE operons. Ler, a master regulator in LEE operons, shares 24% amnio acid identity and 44% amino acid similarity to H-NS. Interestingly, rather than a gene silencer, its main role has been characterized as an antagonizing protein that relieves H-NS-mediated transcriptional silencing. In the previous study we reported molecular mechanism for the repression of LEE5 promoter in EPEC and EHEC by H-NS as a protein interaction between upstream DNA-bound H-NS and the αCTD of promoter-bound RNA polymerase. The mechanism underlying Ler-mediated alleviation of the genes repression by H-NS is largely unknown. We examined regulatory effect of these proteins on LEE5p activity using various in vitro tools. Our results revealed that binding affinity of Ler to the LEE5p DNA is about 40 folds greater than that of H-NS as determined by surface plasmon resonance. We verified that Ler binding removed H-NS bound to the same stretch of DNA on LEE5 promoter resulting in a derepression.