Expressing and secreting two proteins

[Tr0x]

Hello forum! First time posting here but I intend to stick around.

 

I'm wondering (and hoping) if anyone could provide some insight on expressing and secreting two independant proteins in E coli. I've scoured the literature and have found plenty on dual expression (see this article), but nothing that includes secretion too.

 

The idea so far would be to use either a pre-made expression vector like Novagen's Duet and then adding signal sequences in the ORFs to secrete the proteins. Alternatively, I could design my own dual expression vector to do the same thing.

 

I'm hoping to have this ready by September, although I realise it's pretty ambitous considering there's such little info on it. I already have one of the proteins secreting fairly well on its own, and am starting on the second.

 

A stipulation is that I want to be able to continue using cells since I'l eventually be selecting for activity. So the cells must survive the procedure.

 

Ideas?

 

 

[Greippi]

 

The idea so far would be to use either a pre-made expression vector like Novagen's Duet and then adding signal sequences in the ORFs to secrete the proteins.

 

 

That's how I would approach it myself, although I have never done such a thing.

 

As for the cells surviving the procedure - unless I misunderstand what you want to do (which, in retrospect I think I do), when you're doing whatever you'll be doing to purify the protein, only use some of the cells, just leave other cells that are producing your proteins to grow untouched so you have some left over in reserve.

Edited July 23, 2010 by Greippi

[CharonY]

A question I have is whether you want to have a single strain secreting both proteins?

Second is what question you want to answer. Do you want to purify the proteins or assess the function of the proteins in vivo. The latter is a bit more tricky.

[Tr0x]

A question I have is whether you want to have a single strain secreting both proteins?

Second is what question you want to answer. Do you want to purify the proteins or assess the function of the proteins in vivo. The latter is a bit more tricky.

 

Yes, ideally I want to have a single strain secreting both proteins.

 

The question I want to answer is whether I can get "protein 1" to inhibit "protein 2". In other words assessing the function of the proteins in vivo. I already have an assay working to detect functioning "protein 2", but the trick is to get the other one to express and secrete too in the same strain.

 

If it would help, I could provide a bit more detail about the specific proteins, however I don't think it's necessary.

 

Thanks for the help so far!

[CharonY]

This can be quite tricky, depending on the mode of interaction between the proteins you are thinking of. If it is only the isolated protein functions (i.e. no effect on the physiology of the cell) it may be relatively easy (though this would be essentially not different from an in vitro assay). However, if it is supposed to relate to cell activities. it can get complicated.

 

One major issue is their control, i.e. the stochiometric expression of both proteins. Using ordinary overexpression vectors is likely to give odd results. Also if the effects you are expecting are in the extracellular space there is also the issue of the secretion kinetics that may be different from the proteins (even if using identical signal peptides).

Finally there is also the question whether the proteins you are looking at are native in E. coli or whether there may be even more interference. If the proteins in question are known to interact in a specific way and/or e.g. organized in an operon and if they orginated from an organism close to E. coli (including similar regualatory sequences, the simple approach may work. Otherwise I expect issues on several levels. However, it really depends on what you really want to see in the end.

[Tr0x]

Yes, in short it's just the isolated protein functions that I'm interested in. The proteins I'm trying to express are human, meaning they shouldn't have any effect on the e coli colonies, other than reducing their growth rate (which I've observed) as they're making a lot of extra proteins.

 

As for secretion kinetics, I'm aware it's going to be less than ideal but I shouldn't need a whole lot of protein secret to observe it in the assay (fingers crossed that introduces enough).

 

Thanks for the guidance so far! It's great to bounce ideas around. My impression then is that using standard techniques to create a plasmid with two secreting proteins instead of one should do the trick, albeit with a lower yield of each protein.

[CharonY]

Question: if it is not an in vivo test, why not overexpress them, purify them individually and then mix them in defined ratios?

The assay would be much better defined that way.

[Tr0x]

That's a great point! However the assay is meant to screen a whole lot of them easily in order to find the mutant that works. So in order to purify each mutant proteinn from each of the producing colonies will take far too long.

 

I'll probably end up doing that eventually but the first screen is "quick and dirty" in order to sort through all the colonies.

 

Another question: does anyone know of an e coli strain off hand that will be suited to express two recombinant proteins at the same time? I've been using BL21 DE3 so far, and it's worked well for the one protein. The papers I've read regarding this have also used this strain.

[CharonY]

I see. It could be easier to produce strains that express the individual proteins, though, and make cross assays to identify combinations that do something. You will have less problems in producing the strains as well as have less trouble possible cross interactions (e.g. promoter competition) within a strain that may give erroneous results.

[Tr0x]

That's a good point. Having a strain that produces both isn't a necessity; merely a way to avoid having to purify so many possibly useful proteins by looking for interactions while the bacteria were still growing.

 

I'll have to think on this before I decide which way to go. The concept of producing both will be tough to let go of, but the time involved will probably be the same either way.