Eukaryotic recombinant protein expression - (Aug/31/2007 )
Dear all
I have a very basic question about recombinant protein expression:
We have human growth factors intended for therapeutic use, by now we have expressed protein in E.coli for analysis.
Now we think about expressing the proteins also in eukaryotic cells for reason of posttranslational modifications.
This means I would have to clone my sequences in a new plasmid containing a fusion tag, epress the proteine in eukaryotic cells and purify my protein employing the fusion tag afterwards.
After a brief search I did not found to much about this method.
So my question now is how frequently do people use eukaryotic expression for purification of recombinant protein ?
Is it really necessary to worry that much about the posttranslational modifications or is it rather unlikely that expression in E.coli will change the function of human growth factors ?
Please let me know if it is important to do the expression in eukaryotic cells or if its possible to just do it in E.coli.
Also I would appreciate some advice on a good plasmid vector for expression of fusion tagged (His tag favoured) protein in eukaryotic cells.
Thanks
I have a very basic question about recombinant protein expression:
We have human growth factors intended for therapeutic use, by now we have expressed protein in E.coli for analysis.
Now we think about expressing the proteins also in eukaryotic cells for reason of posttranslational modifications.
This means I would have to clone my sequences in a new plasmid containing a fusion tag, epress the proteine in eukaryotic cells and purify my protein employing the fusion tag afterwards.
After a brief search I did not found to much about this method.
So my question now is how frequently do people use eukaryotic expression for purification of recombinant protein ?
Is it really necessary to worry that much about the posttranslational modifications or is it rather unlikely that expression in E.coli will change the function of human growth factors ?
Please let me know if it is important to do the expression in eukaryotic cells or if its possible to just do it in E.coli.
Also I would appreciate some advice on a good plasmid vector for expression of fusion tagged (His tag favoured) protein in eukaryotic cells.
Thanks
Don't know about expressing proteins for therapeutic use. but we have plasmid vector for expression of fusion GST protein in mammalian cells. It works, but we use it for GST pull-down, actually. Suppose you can use for purify protein, but I have never use it for that purpose.
E.coli is a very basic expression system, so it often has trouble expressing eukaryotic genes and certainly can't perform the post-translational modifications that eukaryotes can. When you express these growth factors in E.coli are they soluble and functional? If the E.coli-expressed growth factors are functionally indistinguishable from those expressed in human cells then E.coli should be an acceptable expression host.
If there is a problem with the functional expression in E.coli, then there are many eukaryotic expression systems avaliable. The yeast K.lactis is one example, and is commercially available from NEB. I don't think they have a His-tagging vector but they do have one with a maltose binding protein.
If the k.lactis system is unacceptable, there are many others. Don't be put off if an expression system's vector does not have the HIS tag you require - it is very easy to add your own HIS tag to any vector.
If you are thinking about using these proteins for therapeutic use, you need to think very carefully about using a tag. You are going to have to prove to FDA (or equivalent) that the tag has absolutely no effect. I did a couple of short contracts in a company that expresses human proteins (cytokines and growth factors) in mammalian cells without any tags, because that removes a very significant (read expensive) level of safety testing.
If you are talking about using the protein in an assay, I understand that the current opinion of the function of glycosylation of circulating proteins (as opposed to membrane-bound proteins) is glycosylation provides a supply of ready-to-use proteins that are essentially inactivated. That is, the non-glyco form is the active form, and the glyco form is inactive. So if your system is going to be mainly in vitro, the main benefit of glycosylation is it reduces the frequency of giving the cells the required protein.