Expression systems: Difference between revisions
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= | =Mainstream systems= | ||
==''Escherichia coli''== | |||
===Rosetta cells === | |||
= | If you are trying to express non-bacterial protein, it is quite likely that your gene of interest contains rare codons and thus E.Coli can't express it well. Rosetta and Rosetta 2 cells (available from Novagen) are designed to overcome this problem. For human proteins, expression levels may sometimes change from "I need to enrich the protein batch on affinity column before I can see protein on the gel" to "80% of the protein in the lysed cells is my protein of interest". | ||
'' | |||
==Fungi== | |||
===''Pichia pastoris''=== | |||
==Insect cells== | |||
===Baculovirus-mediated expression in ''Spodoptera frugiperd'' and ''Trichoplusia ni'' cells=== | |||
==Mammalian cells== | |||
= | ===Human Embryonic Kidney (HEK) 293 cultured cells === | ||
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. | |||
= | ===Chinese hamster ovary (CHO) cultured cells=== | ||
=Other systems= | |||
==Bacteria== | |||
===''Bacillus subtilis''=== | |||
===''Bacillus megaterium''=== | |||
===''Pseudomonas fluorescens''=== | |||
==Protozoa== | |||
===''Leishmania tarentolae''=== | |||
==Fungi== | |||
===''Saccharomyces cerevisiae''=== | |||
===''Schizosaccharomyces pombe''=== | |||
===''Hansenula polymorpha''=== | |||
===''Filamentous fungi''=== | |||
=Plant cells= | =Plant cells= | ||
= | ==Algae== | ||
Chloroplasts | |||
''Nicotiana tabacum'' | |||
Latest revision as of 18:34, 12 February 2008
Mainstream systemsEdit
Escherichia coliEdit
Rosetta cellsEdit
If you are trying to express non-bacterial protein, it is quite likely that your gene of interest contains rare codons and thus E.Coli can't express it well. Rosetta and Rosetta 2 cells (available from Novagen) are designed to overcome this problem. For human proteins, expression levels may sometimes change from "I need to enrich the protein batch on affinity column before I can see protein on the gel" to "80% of the protein in the lysed cells is my protein of interest".
FungiEdit
Pichia pastorisEdit
Insect cellsEdit
Baculovirus-mediated expression in Spodoptera frugiperd and Trichoplusia ni cellsEdit
Mammalian cellsEdit
Human Embryonic Kidney (HEK) 293 cultured cellsEdit
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 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 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.
Chinese hamster ovary (CHO) cultured cellsEdit
Other systemsEdit
BacteriaEdit
Bacillus subtilisEdit
Bacillus megateriumEdit
Pseudomonas fluorescensEdit
ProtozoaEdit
Leishmania tarentolaeEdit
FungiEdit
Saccharomyces cerevisiaeEdit
Schizosaccharomyces pombeEdit
Hansenula polymorphaEdit
Filamentous fungiEdit
Plant cellsEdit
AlgaeEdit
Chloroplasts
Nicotiana tabacum