production of mammalian derived proteins and antibodies in weeks
The delivery of DNA into mammalian cells through non-viral methods with temporary effects is known as transient transfection. In the case of delivering functional coding DNA, the term “transient gene expression” (TGE) is widely used. TGE is a commonly used method for the production of recombinant proteins. Whilst commercial production of tons of biopharmaceuticals uses consistently productive stable cell lines (which take many months to develop), the production of milligram to gram amounts of high-grade material for early pre-clinical and biochemical characterization studies can be performed by TGE systems within the drug development pipeline. This website focuses on the production of recombinant proteins and antibodies using TGE. It will give you an overview of the main aspects and influencing factors.
Graphical overview of transient transfection
Target Protein
The target protein can be every imaginable secreted protein. Expression of antibodies in various isoforms, recombinant proteins such as enzymes, virus proteins or cytokines and large fusion proteins are possible. The later usage of the proteins includes, but is not limited to, pre-clinical applications, antigens for immunization, assay development and structural biology.
Secretion
Only correctly folded proteins are transported from the endoplasmic reticulum to the Golgi apparatus, where the target protein is packed into secretory vesicles. These vesicles move toward the cell membrane, fuse, and the target protein is released.
Translation
Generated mRNA leaves the nucleus and can access ribosomes for translation. After the ribosome/mRNA complex binds the membrane of the endoplasmic reticulum, the target protein is synthesized into the lumen.
Transcription
Within the nucleus the gene of interest is transcribed into mRNA by RNA polymerase. For efficient transient gene expression there is the need for high transcription rates. The transcription rate mainly depends, among other factors, on an expression cassette regulated by a strong promotor.
Nucleus internalization
Until now, the detailed mechanism of nucleus internalization is not fully understood. The generally accepted assumption is that the plasmid DNA release is based on polycationic competitors like cytoplasmic RNA or chromosomal DNA. Subsequently, the plasmid DNA enters the nucleus via nuclear pores or during mitosis.
Proton sponge effect
After cellular uptake via endocytosis, the plasmid DNA needs to be protected against lysosomal degradation. Based on available primary and secondary amines within the polyplex, acidification of the lysosome is prevented by proton adsorption. This adsorption leads to an osmolality shift and causes in a water influx bursting the lysosome. This effect is called "proton sponge effect". After polyplex release the plasmid DNA has to enter the nucleus.
Internalization
Since cell membranes are usually negatively charged, positively charged polyplexes interact with the cell membrane and are internalized supposedly through receptors on the cell surface leading in the lysosomal pathway.
Polyplex
Polyplexes are highly condensed positive charged particles formed via electrostatic interactions between the polycationic transfection reagent and the negatively charged plasmid DNA.
Cationic Polymer
Most chemical transfection reagents, especially polycationic polymers, contain a positively charged repetition unit. Usually, the repetition units are derivatives of amino groups.
Plasmid
The plasmid DNA contains the genetic information of your protein of interest. The phosphodiester bonds, which form the DNA backbone, lead to a negatively charged plasmid.
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