How to obtain a non-cellular protein in its natural form? - (Dec/05/2018 )
So I have been having this question for years as I always wondered if the morphology of a protein I express can affect my downstream experiments especially when antibody-anyigen binding such as ELISA is involved.
When a cellular gene is cloned to plasmid and expressed after transfection, most propably it will obtain its natural form and morphology within the cell. Please correect me if I'm wrong.
But not all cloned genes are cellular. Lets say you want to express a viral protein and use it for ELISA. Since the expressed viral protein has not been made through its natural route, i.e. infection and replication, its shape may not be natural either because other paramters or viral proteins are not present. So if you make ELISA and expect a serum to react with this protein you may be dissapointed. That said, I have seen many articles and protocols for bacterial or viral ELISA production that simply follow the plasmid expression method without any problem.
But the other day I came across GenScript's protein synthesis service for up to 200 aa peptide. They can do lots of modification and sort of customize your protein including disufide bridge.
So I was thinking if your viral peptide or protein of interest is short then why not just chemically syntheize and modify it according to the shapes available in the PDB database to make it look as natural as possible. This way you dont even have to try to design primers, clone, transform, extract, transfect, lyse and purify. You will skip this long procedure.
Does anybody know if such approach is possible? Does anybody do it that way? Can you share articles with me please?
Just a thought: Even though a viral protein is not encoded by a cell, it is still expressed by a cell, since viruses are obligate intracellular parasites. They have evolved specifically to express their proteins through the protein synthesis machinery of host cells, and the viral proteins gain their folded architecture within the cell.
As for synthetic proteins, if you know what its crystal structure should be, and they can make it that way, it should potentially work. If it can be assembled in its mature form- some proteins having multiple peptides that must be put together. This of course would be a great science experiment, to see if complicated synthetic proteins behave like the natural ones. Obviously the companies offering the synthesis think so.
It is an interesting question, and one that has also been wandering around in my head for a while. We know that many proteins (e.g. restriction enzymes) express well and still function perfectly as expected when purified and concentrated. This is not necessarily the case for many proteins though - particularly those that are part of a larger complex, or are large proteins. I would be willing to bet that the crystal structures of many proteins are not a complete active form - active forms are likely to require minor morphological shifts in active sites etc.
If you log on to GenScript, Thermo or SIGMA websites they offer many modifications to your protein of interest. I was actually very surprised while I was filling the online forms. As someone who does not usually order synthetic proteins, the modifications and labels were confusing. I had not heard those terms and names in my life. So I told myself if so many modifications are available it means that these companies have many customers. Perhaps they get many orders for complicated structures every day. That is why the technology has rapidly improved over the years.
Just now a question came to my mind. If you express a protein that has two cysteine residues, do they naturally get disulfide bonds between them? or an enzyme needs to be present?
Curtis on Fri Dec 7 10:13:33 2018 said:
Just now a question came to my mind. If you express a protein that has two cysteine residues, do they naturally get disulfide bonds between them? or an enzyme needs to be present?
I think it si a folding issue. The cysteines have to be in certain distance and while the peptide is formed on mitochondria/ER it gets folded (and maybe some general chaperones are present in the cell enviroment), but AFAIK the bond formation is automatic when those C fold in close proximity.