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Expression systems: Difference between revisions
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→Human Embryonic Kidney (HEK) 293 cultured cells
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===Human Embryonic Kidney (HEK) 293 cultured cells === | ===Human Embryonic Kidney (HEK) 293 cultured cells === | ||
Commonly | Commonly known as HEK293 cells they are actually an excellent protein source, especially for secreted proteins. Contrary to urban legend, they are significantly cheaper than insect cells to culture. You need standard cell culture equipment that in most cases is available in any biology department. Cloning is easy and a wealth of vectors are available to insert your gene in. contrary to insect cells, the DNA can be used directly for | ||
[http://www.ncbi.nlm.nih.gov/pubmed/17001101?ordinalpos=6&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum transient transfections]. | |||
In some cases it is worth investing in generating a stable cell line that produces the protein of interest; a good tool to do that is the | |||
[http://www.invitrogen.com/content.cfm?pageid=3375 Flp-In system from Invitrogen ] that can be used to generate highly expressing stable cell lines. | |||
Quite easy to scale up the volume still using attached cells, in special plastic bottles that present a large surface area: thats most useful especially for secreted proteins. There are two main ways to express your gene in HEK293 cells: | |||
Producing intracellular proteins in HEK293 cells requires to grow the cells in suspension which can be a bit more of an art and requires a CO2 shaking incubator. | Producing intracellular proteins in HEK293 cells requires to grow the cells in suspension which can be a bit more of an art and requires a CO2 shaking incubator. | ||