Mammalian expression system into a cell can be transient or stable. stable expression system can refer to a cell line that is simply stable, in that it will remain unchanged over a long time. Not a common usage of the term. A cell line by definition is pretty stable, at least most are. It can refer to a cell line that has been transfected or infected with a foreign piece of DNA that has incorporated itself into the genome of the cell.
In stable expression system, the plasmid DNA is integrated into the chromosomes, or as an episome a separate piece of nuclear DNA), and gets passed on to future generations of the cell. This is a much rarer occurrence and complex to perform, as sometimes only a portion of the plasmid gets integrated, which may not contain the gene of interest. All stable expression system transfections start out as a transient transfection, with selectable markers that are able to distinguish any cells that have successfully integrated the gene into their genome. A common method used is to co-transfect the gene of interest with another gene for antibiotic resistance, and treat the transfected cells with the antibiotic. Repeating antibiotic treatment for long-term cell cultures results in the expansion of stably-transfected cells. Linear DNA appears to be better for stable expression system, although its uptake is lower than supercoiled DNA.
Mammalian expression system are generally capable of producing larger amounts of properly folded secreted proteins with usually correct glycosylation pattern for mammalian or other eukaryotic proteins. In case of cytoplasmic proteins or multi-protein complexes, mammlian expression system show usually lower expression levels than insect cells which are also capable of secreting proteins if a native-like glycosylation pattern is not essential for the functionality of the target protein or the planned down-stream application. Mammalian cells expression system covering transient expression system and stable cell line expression system.
Transiently transfected cells express the foreign gene but do not integrate it into their genome. Thus the new gene will not be replicated. These mammalian cells express the transiently transfected gene for a finite period of time, usually several days, after which the foreign gene is lost through cell division or other factors.
Generating stably transfected cells begins with a transient transfection, followed by an infrequent but important and serendipitous process. In a small proportion of transfected cells, the foreign gene is integrated into the cells’ genome. The hallmark of stably transfected cells is that the foreign gene becomes part of the genome and is therefore replicated. Descendants of these transfected cells, therefore, will also express the new gene, resulting in a stably transfected cell line.
As the development of stably-expressing cell lines can be laborious and challenging, the ability to use transient expression when appropriate is beneficial. Along with new transfection techniques will undoubtedly come the further refinement of both transient and stable transfection methods, enabling researchers to accomplish gene expression tasks as efficiently as possible.
Mammalian expression system category from:
Most energy-conserving reactions in living organisms are redox reactions. One substrate is oxidized with the concomitant reduction of another substrate. In chemoorganotrophic aerobes, the substrate reduced is usually oxygen. In respiring anaerobes, the electron acceptor can be either organic or inorganic. Typical examples are the sulfate-reducing or methanogenic organisms (carbon dioxide). In respiring organisms, both aerobic and anaerobic, most of the energy is produced by electron transport phosphorylation. This is in contrast to fermentations, in which most of the adenosine triphosphate (ATP) is synthesized by substrate level phosphorylation.
Fermentation is an anaerobic redox process, in which the oxidation of the substrate is coupled to the reduction of another substrate or an intermediate derived from the oxidation, with the difference in redox potential of the substrate and the end product providing energy for ATP synthesis. In most fermentations, the same substrate is used as both reductant and oxidant, whereas in some amino acid fermenting organisms, one amino acid is oxidized and another is reduced (Stickland reaction). The oxidation reaction is coupled to substrate level phosphorylation whereas the reduction reaction is usually not. The fermentation end products are excreted. The nature of these products is different in various species, and the various fermentation pathways are named after their main products.
This is a 5-day protocol for high cell-density bacterial fermentation and overnight over-expression of (a) target protein(s) using a computer controlled fed-batch procedure.
|Protocol in short|
|Day 1||Assembly of the fermenter vessel Preparation and autoclavation of the fermenter vessel
Preparation of the starter culture
|Day 2|| Preparation of inoculation cultures Preparation of the fermenter vessel for cultivation
Inoculation of the fermenter vessel and start of the batch phase
|Day 3||Start of the fed-batch phase
Induction of protein expression
|Day 4|| Harvesting of the cells
Cleaning of the fermenter vessel
More details for experimental steps: High cell-density fermentation of Escherichia coli
The BIC(BiologicsCorp) bioprocess facility provides services in microbial fermentation. It features a 1,000 sq. ft. pilot scale fermentation laboratory capable of handling class II organisms, an adjoining 500 sq. ft. bench scale cell culture, and fermentation laboratory with multiple fermenters and bioreactors for microbial. The facility can manufacture recombinant proteins and metabolites, as well as media optimization and cell culture scalability experiments. The fermentation facility provides clients with seed culture development and culture scale-up from shake flasks to pilot scale fermenters.
BIC(BiologicsCorp) has a number of 5L, 50L, 100L, and 1000L fermentors available on-site for fermentation runs. BIC’s fermentation services include: