Abstract
Gene expression in transduced mammalian cells correlates with virus titer, but high doses of vector for gene therapy leads to toxicity in humans and in animals. Changing the optimal tissue culture medium by adding low levels of environmental stressors, such as 1 µM of the fungal toxin aflatoxin B1 (AFB1), 1 ng of the castor bean protein toxin ricin, or 1% reconstituted milk, enhances transcription and increases production of proteins in transduced mammalian cells as demonstrated by production of the following three recombinant proteins: firefly luciferase, β-galactosidase, and green fluorescent protein (GFP). Higher concentrations of the stress-producing substances damage the cells beyond recovery, resulting in inhibited gene expression and cell death. We also evaluated the effect of the stressor substances on the enhanced infectivity of virus. The presented findings extend methods for large-scale transient recombinant protein production in mammalian cells and suggest that it may be possible to reduce the cytotoxicity of the adenovirus by reducing the virus titer without adversely affecting gene expression levels.
Highlights
Therapeutic recombinant protein expression is widely used in medicine and agriculture [1]
We demonstrated that changing the optimal tissue culture medium with added low concentrations of milk or the known toxins aflatoxin B1 (AFB1) and ricin results in enhanced recombinant protein production, as demonstrated by production of three recombinant proteins tested in transduced mammalian cells
Low concentrations of plant and fungal toxins induce recombinant gene expression We determined that changing the optimal cell culture medium by adding increasing concentrations of the plant toxin ricin or the fungal toxin AFB1 would enhance the expression of recombinant green fluorescent protein (GFP) production in Vero cells transduced with adenovirus-green fluorescent protein (Ad-GFP) following 48 h incubation
Summary
Therapeutic recombinant protein expression is widely used in medicine and agriculture [1]. About 60% of all recombinant pharmaceutical proteins are produced in mammalian cells, mainly because mammalian cells can post-translationally modify proteins, and thereby significantly enhancing protein bioactivity. It seems that the expression levels in mammalian cells are generally lower than in bacterial systems. There is a need to increase the yield of recombinant proteins expressed in mammalian cells, and one objective of the present study is to meet this need. In this study we used adenovirus as a vector, widely used in gene therapy, to introduce recombinant DNA to mammalian cells. A further objective of this study was to minimize the cytotoxic effects of the virus by reducing the virus titer without reducing the gene expression levels
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
