Publication

A simple plasmid-based transient gene expression method using High Five cells

Florian Maria Wurm, David Hacker, Lucia Baldi Unser, Virginie Bachmann, Xiao Shen
2015
Journal Articles

Abstract

The High Five (H5) cell line, derived from the lepidopteran Trichoplusia ni, is one of the major insect cell hosts for the production of recombinant proteins using the baculovirus expression vector system (BEVS). Here, we describe a simple polyethylenimine (PEI)-based transient gene expression (TGE) process for the rapid production of recombinant proteins from suspension-adapted H5 cells. The method was optimized using two model proteins, enhanced green fluorescent protein (EGFP) and human tumor necrosis factor receptor-Fc fusion protein (TNER-Fc). After screening several promoter and enhancer combinations for high levels of TNER:Fc production, an expression vector containing the Autographa californica multicapsid nucleopolyhedrovirus immediate early 1 (ie1) promoter and homologous region 5 (hr5) enhancer was selected. Cells were transfected at a density of 2 x 10(6) cells/mL by direct addition of DNA and PEI. Under optimized conditions, a 90% transfection efficiency (percentage of EGFP-positive cells) was obtained. In addition, we observed volumetric TNER-Fc yields over 150 mu g/mL within 4 days of transfection. The method was found to be reproducible and scalable to 300 mL. This plasmid-based transient transfection process is a simple and efficient alternative to the BEVS for recombinant protein production in H5 cells. (C) 2015 Elsevier B.V. All rights reserved.

Official source

https://infoscience.epfl.ch/entities/publication/8e716b7d-35cb-4e94-8a9d-08f8496e4b07

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Florian Maria Wurm, David Hacker, Lucia Baldi Unser, Virginie Bachmann, Xiao Shen
2015
Journal Articles

Abstract

Official source

https://infoscience.epfl.ch/entities/publication/8e716b7d-35cb-4e94-8a9d-08f8496e4b07

About this result

Related concepts (29)

Protein production

Protein production is the biotechnological process of generating a specific protein. It is typically achieved by the manipulation of gene expression in an organism such that it expresses large amounts of a recombinant gene. This includes the transcription of the recombinant DNA to messenger RNA (mRNA), the translation of mRNA into polypeptide chains, which are ultimately folded into functional proteins and may be targeted to specific subcellular or extracellular locations.

Expression vector

An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. Expression vectors are the basic tools in biotechnology for the production of proteins. The vector is engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector.

Cell culture

Cell culture or tissue culture is the process by which cells are grown under controlled conditions, generally outside of their natural environment. The term "tissue culture" was coined by American pathologist Montrose Thomas Burrows. This technique is also called micropropagation. After the cells of interest have been isolated from living tissue, they can subsequently be maintained under carefully controlled conditions. They need to be kept at body temperature (37 °C) in an incubator.

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