Laboratory production of recombinant mammalian proteins, particularly antibodies, requires an expression pipeline assuring sufficient yield and correct folding with appropriate posttranslational modifications. Transient gene expression (TGE) in the suspension-adapted Chinese Hamster Ovary (CHO) cell lines has become the method of choice for this task. The antibodies can be secreted into the media, which facilitates subsequent purification, and can be glycosylated. However, in general, protein production in CHO cells is expensive and may provide variable outcomes, namely in laboratories without previous experience. While achievable yields may be influenced by the nucleotide sequence, there are other aspects of the process which offer space for optimization, like gene delivery method, cultivation process or expression plasmid design. Polyethylenimine (PEI)-mediated gene delivery is frequently employed as a low-cost alternative to liposome-based methods. In this work, we are proposing a TGE platform for universal medium-scale production of antibodies and other proteins in CHO cells, with a novel expression vector allowing fast and flexible cloning of new genes and secretion of translated proteins. The production cost has been further reduced using recyclable labware. Nine days after transfection, we routinely obtain milligrams of antibody Fabs or human lactoferrin in a 25 mL culture volume. Potential of the platform is established based on the production and crystallization of antibody Fabs and their complexes.

• Klíčová slova
• CHO cell line, gene delivery, polyethylenimine, production costs, protein crystallography, recombinant protein production,
• Publikační typ
• časopisecké články MeSH

A large number of signalling pathways converge on p53 to induce different cellular stress responses that aim to promote cell cycle arrest and repair or, if the damage is too severe, to induce irreversible senescence or apoptosis. The differentiation of p53 activity towards specific cellular outcomes is tightly regulated via a hierarchical order of post-translational modifications and regulated protein-protein interactions. The mechanisms governing these processes provide a model for how cells optimize the genetic information for maximal diversity. The p53 mRNA also plays a role in this process and this review aims to illustrate how protein and RNA interactions throughout the p53 mRNA in response to different signalling pathways control RNA stability, translation efficiency or alternative initiation of translation. We also describe how a p53 mRNA platform shows riboswitch-like features and controls the rate of p53 synthesis, protein stability and modifications of the nascent p53 protein. A single cancer-derived synonymous mutation disrupts the folding of this platform and prevents p53 activation following DNA damage. The role of the p53 mRNA as a target for signalling pathways illustrates how mRNA sequences have co-evolved with the function of the encoded protein and sheds new light on the information hidden within mRNAs.

• MeSH
• 3' nepřekládaná oblast genetika   MeSH
• 5' nepřekládaná oblast genetika   MeSH
• fyziologický stres genetika   MeSH
• lidé MeSH
• ligandy MeSH
• messenger RNA genetika   MeSH
• nádorový supresorový protein p53 genetika   MeSH
• protoonkogenní proteiny c-mdm2 metabolismus   MeSH
• riboswitch genetika   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• 3' nepřekládaná oblast MeSH
• 5' nepřekládaná oblast MeSH
• ligandy MeSH
• messenger RNA MeSH
• nádorový supresorový protein p53 MeSH
• protoonkogenní proteiny c-mdm2 MeSH
• riboswitch MeSH

The biological fate of each mRNA and consequently, the protein to be synthesised, is highly dependent on the nature of the 3' untranslated region. Despite its non-coding character, the 3' UTR may affect the final mRNA stability, the localisation, the export from the nucleus and the translation efficiency. The conserved regulatory sequences within 3' UTRs and the specific elements binding to them enable gene expression control at the posttranscriptional level and all these processes reflect the actual state of the cell including proliferation, differentiation, cellular stress or tumourigenesis. Through this article, we briefly outline how the alterations in the establishment and final architecture of 3' UTRs may contribute to the development of various disorders in humans.

• MeSH
• 3' nepřekládaná oblast MeSH
• chorea genetika   metabolismus   patologie   MeSH
• dědičné degenerativní poruchy nervového systému genetika   metabolismus   patologie   MeSH
• demence genetika   metabolismus   patologie   MeSH
• expanze trinukleotidových repetic MeSH
• kognitivní poruchy genetika   metabolismus   patologie   MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• myotonická dystrofie genetika   metabolismus   patologie   MeSH
• nádory genetika   metabolismus   patologie   MeSH
• prekurzory RNA genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• 3' nepřekládaná oblast MeSH
• prekurzory RNA MeSH