So now that I have finished writing the BSF grant, I am finally free to get back to the bench and start kicking some serious work. One of my “on the shelf proteins” is a small protein less than 20 kDa which expresses poorly – so, in this post I will discuss how to boost recombinant protein expression in E. Coli.
For example, see below a comparison of coomassie staining and blotting with anti-his antibody:
Now, when I see such low expression (after verification that this is indeed my protein), I ultimately know that I have a long way till I will start any serious purification process. On the other hand, if I see such a sight as the one below, I will quickly arrange myself an FPLC for the next week:
That’s a good sign! Highly induced protein expression (lane 2) as seen under coomassie staining
So, how can I improve the expression? How can I get that thin band turned into a fatty??
Well, first of all let’s consider what IS a fatty band on a SDS-PAGE?
A fatty is a protein which is highly/easily expressed by the bacteria at the allocated expression setup (time and temperature) AND is relative stable AND folds correctly AND is not (highly) degraded by the proteolytic plethora within E.Coli AND doesn’t lead to cytotoxicity. As you see, there’s a lot of AND requirements to get a fatty, and this is just the intuitive list. The highlighted part is the one part that comprise of most factors that dearly affects the end result of your expression. So, I will start with that and then move on to the other parts, which are no less important.
The expression stage is the rate-limiting step and it is the first factor that should be checked off when encountering a though-cookie-protein. The factors that lead to an optimal expression are among others:
- Plasmid copy – It is known that overtime bacteria can lose the expression vector. Use fresh transformants cells.
- Intact Promotor – Mutations at the promotor or at the RBS can really make expression a waste of time.
- Check for rare codon usage (see https://www.genscript.com/cgi-bin/tools/rare_codon_analysis and http://nihserver.mbi.ucla.edu/RACC/).
- Time & temperature – Don’t stick to the common paradigm (i.e., grow at 37 degrees till O.D=0.6, then add 0.1-1mM IPTG and grow for additional 3 hours). Try different temperatures (from as low as 16 degrees to 30 degrees) and for varying times (as long as a whole weekend!).
- Media – Try autoinduction media (Sutdier, 2005) – expression level can be easily bumped up to ten fold!
Stability and fold
If nothing of the above has worked, it’s time to evaluate whether the protein expresses but is not folded correctly or whether it is expressed and folded but quickly degraded by the host proteases. In most cases of instable protein some of the expressed will be still visible and possibly also it’s degradation products (for both of these you will need to use western blotting for the identification of low level detection). In the case of a suspected protease-driven degradation, try to search for proteolytic sites within your protein’s sequence. If you find such sites, there is no other option but to mutate the recognition site.
You might find the protein expresses well but most of it is in the pellet fraction. While this mostly is the case with membrane proteins, it can be the case with proteins which unfolds correctly or which have low solubility. Incorrect fold can be overcome with the use of chaperon co-induction (see kit from Takara) while low solubility can be overcome with the use of MBP/GST-fused protein.
Toxicity proteins can be detected as you will see your inoculum not growing to high O.D. as culture growth will be arrested at a certain point. The inoculum might eventually reach high O.D. but it might be due to loss of the plasmid containing the resistance gene (especially with Ampicilin resistance gene). Dealing with toxic protein should be performed by a combination of low induction temperature (slowing down translation) and utilizing the pLysS strain which contains T7-Lyzozyme, a naturally inhibitor of T7-RNA polymerase and thus eliminating any basal expression from the T7 promotor.
What to do when all the above don’t increase yield??
If you get to the point when nothing of the above solutions helps you get a nice expression, you might consider chopping off some of the protein’s flanking areas and focusing on a certain domain. This strategy might not fit all biochemist, which in many cases aim at characterization of the whole protein, but it can make the difference between a low to no expressed protein to a highly expressed and (!) characterized protein.
You might want to have a look at these excellent web resources: