Hi Guys, hope all is well with you. I want to overexpress a protein using HEK 293 Cell line. Could you please kindly tell me what is the most convenient way to do so. I read something about finding a clone for the protein gene, could you kindly clarify on how to do so. Many thanks for your time.
Hi Moe,
You start with a plasmid that contains cDNA of the protein you want to over express. Some of cDNA are quite cheap but some can be more than 2k dollars. Sometimes you will have to clone out your cDNA of interest.
Once you have plasmid with the clone, you need to transfect cells. Hek293 are easy to transfect, I use lipofectamin or prime jet reagent. After this you need to do antibiotic selection in order to generate a new cell line that will over express the protein of interest and characterize it. Whole process takes aprox.. 2 months.
hope this helps. If you need details about any particular step I might be able to provide you with a protocol.
Hi:
1. Firstly clone the cDNA of interest OR buy in clone (see sites like IMAGE clone)
2. Put clone in to suitable plasmid for mammalian expression (i.e. CMV promoter etc)
3. At this stage I would only look at transient transfection on adherent cells to see if your clone expresses and for this you can use a number of different methods: Mirus/Lipofectamine/PEI etc. The main purpose though is to see if it expresses and is processed as you would expect i.e. size etc. you may also be able to use sample from this for initial testing i.e. bioactivity. At first his can be small scale 6-24 well plates. If you have an Elisa or other methods of quantification you can estimate the levels of protein obtained.
4. Depending on how much protein you require (this is important and should be decided in advance) you may wish to produce a stable cell line or scale up transient transfections (see Durocher Y etal for good transfection advice). Generally we use a suspension adapted cell line.
We use a CHO Flp-In system (invitrogen) for a quick generation of stable clones (~2 wks) but also use transient transfection with PEI (Hek/CHO). There are many papers out there describing stable clone generation but it may not be necessary for you to go through some methods that take months to complete: A simple stable transfection and quick selection with antibiotics would give you a pool of stable transfectants in a couple of weeks and may generate sufficient material for testing.
5. Generally depending on amounts of protein you will more than likely have to express you protein from a suspension adapted/protein free culture: we use Hyclone media from thermo for CHO. There are cell lines available Hek293F cells (invitrogen) that are already adapted to suspension culture.
hope this helps
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