Tuning plasmid DNA amounts for cost-effective transfections of mammalian cells : when less is more
Carreño, Aida (Universitat Autònoma de Barcelona. Institut de Biotecnologia i de Biomedicina "Vicent Villar Palasí")
Guerrero-Yagüe, Rubén (Universitat Autònoma de Barcelona. Institut de Biotecnologia i de Biomedicina "Vicent Villar Palasí")
Casal, Enriqueta (Universitat Autònoma de Barcelona. Institut de Biotecnologia i de Biomedicina "Vicent Villar Palasí")
Mendoza, Rosa (Universitat Autònoma de Barcelona. Institut de Biotecnologia i de Biomedicina "Vicent Villar Palasí")
Corchero Nieto, José Luis (Universitat Autònoma de Barcelona. Departament de Genètica i de Microbiologia)

Date:	2024
Abstract:	Transient gene expression (TGE) in mammalian cells is a well-known approach to the fast expression of recombinant proteins. The human cell line HEK (human embryonic kidney) 293F is widely used in this field, due to its adaptability to grow in suspension to high cell densities in serum-free media, amenability to transfection, and production of recombinant proteins in satisfactory quantities for functional and structural analysis. Amounts of plasmid DNA (pDNA) required in transfections for TGE remain high (usually 1 µg pDNA/mL, or even higher), representing a noticeable proportion of the overall cost. Thus, there is an economic need to reduce amounts of coding pDNA in TGE processes. In this work, amounts of both pDNA and transfecting agent used for TGE in HEK 293F cells have been explored in order to reduce them without compromising (or even improving) the productivity of the process in terms of protein yield. In our hands, minimal polyethyleneimine (PEI) cytotoxicity and optimum protein yields were obtained when transfecting at 0. 5 µg pDNA/mL (equal to 0. 5 µg pDNA/million cells) and a DNA-to-PEI ratio of 1:3, a trend confirmed for several unrelated recombinant proteins. Thus, carefully tuning pDNA and transfecting agent amounts not only reduces the economic costs but also results in higher recombinant protein yields. These results surely have a direct application and interest for the biopharmaceutical industry, always concerned in increasing productivity while decreasing economic costs. • Mammalian cells are widely used to produce recombinant proteins in short times. • Tuning DNA and transfecting agent are of great interest to optimize economic costs. • Reducing DNA and transfecting agent amounts result in higher protein yields.
Grants:	Instituto de Salud Carlos III PI20/00770 Instituto de Salud Carlos III PI17/00553 Instituto de Salud Carlos III PI17/00150 Instituto de Salud Carlos III PI20/00623 Ministerio de Ciencia e Innovación PRE2019-089017
Note:	Altres ajuts: acords transformatius de la UAB
Rights:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Language:	Anglès
Document:	Article ; recerca ; Versió publicada
Subject:	Polyethylenimine ; Transfection ; Transient gene expression ; HEK 293F cells ; Recombinant protein
Published in:	Applied Microbiology and Biotechnology, Vol. 108 (January 2024) , art. 98, ISSN 1432-0614

DOI: 10.1007/s00253-024-13003-x
PMID: 38212965

11 p, 2.2 MB

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Research literature > UAB research groups literature > Research Centres and Groups (research output) > Health sciences and biosciences > Institut de Biotecnologia i de Biomedicina (IBB)
Articles > Research articles
Articles > Published articles