[Lii]

I still have so much trouble understanding proteins.

Basically, I need to design a strategy through which I can express my given protein. I need to make choices about expression vector, tags, host organism and the like. Right now my issue is... I know how expression is done, I've just never thought about how people decide what organisms to use.

My protein is from a bacterium and I know its sequence. I also ran a secondary structure prediction, so I know the locations of buried/exposed regions and disordered regions and the like. I have also run stability predictions and I have quite a bit of other info about this protein.

Now I'm just having trouble making the jump from what I have to what vectors/hosts/etc I should use.

I've heard that for proteins from prokaryotes, the obvious choice is to use E. coli. Is that a good idea?

Any other ideas for what I should start looking into first?

[Yggdrasil]

Here's an incredibly useful paper for those looking to do recombinant protein purification, but have not done so before:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3178102/

E. coli is the best organism to star with because it's cheap to use and often will give high yields.  If you're working with a large, multi-domain eukaryotic protein, bacterial overexpression will often fail, in which case one would try a different expression system (e.g. yeast or baculovirus).  But, trying to express your protein in bacteria is always a good first step.

[Babcock_Hall]

That looks like a very good reference.  I have also used high-expression plasmids that were based upon the trc or tac promoters.  It might help to list out the properties of a high-expression plasmid and check them off when you encounter a new one.

[Lii]

That paper is wonderful! Thank you!

I read that E. coli doesn't do well with disulfide bonds. My protein only contains one, so do you think it would still be okay? It sounded like there would just be an issue if there were multiple disulfide bonds.

[Yggdrasil]

Yes, proteins that require disulfide bonds are one class of protein that can be difficult to express in bacteria.  I don't have much experience trying to express these types of proteins in bacteria, but here's some advice I can give:

1) Is the disulfide bond actually necessary?  If you are able to do genetic experiments in the organism from which the protein originates, does mutating one or both of the cysteines to serine affect the in vivo function of the protein.  If not, you may be able to get away with expressing and purifying the mutant.

2) Proteins that require disulfide bonds for correct folding often misfold in bacteria and form aggregates called inclusion bodies.  If you protein is a small, single domain protein, you may want to try purifying these inclusion bodies, denaturing them, then refolding in the presence of oxidizing agents to obtain your protein.  This can work quite well in many cases and I've had a lot of success with this approach (it often yields a much cleaner protein prep as the inclusion bodies basically contain only your protein of interest).  Here's a useful reference for inclusion body purification http://www.sciencedirect.com/science/article/pii/S0076687909630172

3)  You could try expressing your protein in the periplasm of E. coli or using a strain engineered to facilitate disulfide formation in the cytoplasm.  Here's a paper discussion these strategies: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2689190/

As always, the optimal strategy will depend on the particular details of your protein and the applications for which you'd like to use the purified protein.  Finding an appropriate expression strategy will often require trying a few approaches until you find one that works.