I have been trying to over-express my protein in bacteria but in vain. Alternatively, is it possible to isolate the protein from cell lines/tissues using an antibody and then purify the protein and use it for other downstream experiments which could include deciphering the structure, etc.?
Suggestions and protocols would be highly appreciated.
theoretically you can do that but there are going to be several practical issues. how are you going to elute your protein? Most likely by using extreme ph buffer. How are you going to ensure your protein is still folded? Are you using tagged protein? Then are you going to elute your protein with the peptide? You need milligrams quantity of protein for its structural characterization. How much money you want to spend on the resin and/or peptide? are you going to purify native protein(without over expression)? if yes, what is abundance of your protein in those cell lines/tissue?
These are some of the practical challenges you are going to face.
@Dhiraj Srivastava thanks for the suggestions. I guess I'll have to re-frame my experiments again. But yeah thanks a ton.
Dear Rohit
i do not know the nature of your protein (it is a membrane protein?) and the kind and amount of trial that you performed by e.coli (e.g use fusion tags) but before think to cell lisates i will of course think on recombinant expression on mammalian cells by transient transfection. i'm using expi293 system and it is expensive butreally powerfull. of course you can habe some issue related ti glycosilation that can make more difficult your crystallization but this issue will be also present by extraction from cell lysate where the expression level is very low and if you would like to purify by antibody as dhiraj suggest, you need to use extreme condictions for separate the protein and the antibidy that can cause protein unfolding.
good luck
Manuele
Manuele Martinelli yep !! My protein is cytoplasmic. I am thinking of reframing my experiments and re-attempting induction trials after going through what Dhiraj Srivastava says !!
As Manuele Martinelli suggested, you can consider recombinant expression in cell line s like HEK or gnt cells. while transient transfection gives better yield and fast to begin with, its neither time nor cost effective when you are going to scale it up. For large scale and repetitive expression and purification, you may want to make stable cell lines. It may have poorer expression but you dont need to transfect large amount of cells, everytime you are going to expresse your protein saving both your time and expensive transfection reagent.
you might want to try gnt-1 cells that has reduced glycosylation. You can further remove glycosylation by endoH treatment.
Please note that none of the two are going to be perfect for removing glycosylation and its going to be difficult to crystallize.
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