KMD Bioscience Co., Ltd.
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2020-10-28 Hits(1074)
Mammalian cells can correctly and effectively express eukaryotic proteins, including the synthesis, processing, and secretion of proteins. The mammalian system has the excellent function of post-translational modification of proteins and can assemble proteins correctly. This is because mammalian systems have endoplasmic reticulum, Golgi apparatus and other organelles that are key elements in cell transport and post-translational modification.
Most post translational modifications occur during or shortly after translation. Excreted proteins are made in the endoplasmic reticulum where there are enzymes that facilitate proper folding, disulfide bonding, and N-linked glycosylation. Additionally, before it is excreted, a protein would pass through the golgi apparatus, where O-linked glycosylation may occur as well. All of these modifications occur in many proteins expressed by mammalian cells, including antibodies.
However, the expression level of the mammalian system is low, the medium component is complex, and the experimental cost is relatively high, so it is challenging to produce a large number of proteins.
1. Experimental Process
●Gene synthesis and plasmid construction: the target gene is connected to vector by the method of double enzyme digestion, and its correctness is tested by enzyme digestion and sequencing.
●Plasmid transformation and max-prep: plasmid transformation competent cells & transfected plasmid max-preparation.
●Cell transfection: method of expression transfection for the mammalian cell are transient transfection and stable transfection expressions.
●Cell culture and protein expression.
●Protein purification & QC
2. Transient Transfection
The cells frequently used for transient transfection are HEK293 or CHO cells. Transiently transfected cells express the foreign gene but do not integrate it into their genome. Thus the new gene will not be replicated. The effects of the foreign gene within the cell last only a short amount of time (usually several days), after which the foreign gene is lost through cell division or other factors.
The plasmid DNA (or other type of nucleic acid) typically has a reporter gene that allows a scientist to monitor the expression of the reporter gene, usually within 1-2 days post-transfection.
3. Stable Transfection
Generating stably transfected cells begins with a transient transfection. In a small proportion of transfected cells, the plasmid DNA successfully integrates into the cellular genome and will be passed on to future generations of the cell.
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 expressed cell line. Stable cell lines can grow continuously over a long period of time without significant change in the expression level of the target gene.
A common method used is to design the plasmid DNA to also contain a gene that expresses antibiotic resistance. Continued antibiotic treatment of the cells for long-term results in the expansion of only the stably-transfected cells, while non-stable cells die off due to the lack of antibiotic resistance.
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