Part 1:
Improving protein yield using T7 Express and NEB Express E.coli expression systems
Successful protein expression and isolation are prerequisite for many downstream applications, such as protein crystallization and functional assays. While there exists a myriad tools for protein expression, most of which work well for simple, non-toxic, cytosolic proteins, many other peptides e.g. membrane proteins present greater challenges and the purpose of this blog post series is to help you understand what tools are available to overcome these.
In this first instalment we are troubleshooting issues with low yield in E. coli based expression systems.
What host strain should I use?
The first question one has to answer is what host strain best suits the experiment. Engineered E. coli strains are normally specialized for either protein expression or cloning, although some, such as T7 Express and NEB Express, carry a mutation of the endA1 gene which results in abolished Endonuclease I activity, and thus provide favourable conditions for both cloning and protein expression in a single strain.
Transformation was successful but my cells aren’t growing well, what can be the problem?
If you are trying to express a protein under the control of a strong promoter, particularly if the protein of interest is heterologous, constitutive low level synthesis of the product may cause issues with host cell viability through multiple pathways (e.g. cytotoxicity, discussed below).
Issues with “leaky” expression can be circumvented by utilizing an inducible promoter system. An example of such a system employs promoters responsive to Isopropyl β-D-1-thiogalactopyranoside (IPTG), however these may require additional regulation to completely avoid basal expression. This can be achieved by the inclusion of a repressor, for instance LacI (or similar)-encoding gene for systems based on Lac-promoter variants. There are several commercially available strains which include the enhanced laclq – gene which results in a ten-fold higher expression of the LacI represor, e.g NEBExpress® Iq.
As an option to ITPG-inducible promoters in IPTG-independent expression systems, such as T7, control of expression can instead be achieved by co-expression of T7 lysozyme, which interacts with and inhibits the function of the T7 polymerase. Several variants of T7 lysozyme are widely used depending on the application (more details here) and can be combined to tailor function to the needs of the speficic expression.
What if my cell culture grows fine but I’m still getting low protein yield?
There are of course many possible reasons for this, a common one being that the host strain produces proteases, which successively degrades your protein product. The go-to solution is to use host strains where protease activity has been knocked down, or out, as is the case in several commercially available strains. In cases where some protease activity must be retained during synthesis, addition of a protease inhibitor cocktail during the initial protein purification steps can help increase yield.
What can I do if I suspect my protein of interest to be toxic?
Effective and precise tuning of the expression of your protein of interest is even more vital when the protein product risks becoming toxic to the host cell, as it can allow the user to keep protein levels low enough to avoid toxicity, thereby maximizing yield. The same method can also be used to prevent formation of inclusion bodies when expressing otherwise non-toxic proteins.
One way of doing this is by tightly controlling T7 Lyzosome Y (LysY) expression using a L-rhamnose-dependent promoter, PrhaBAD, resulting in expression of the protein of interest in a manner inversely proportional to the supplied rhamnose concentration. Once again, our supplier New England Biolabs, has a tool for this in the form of Lemo21(DE3) competent E. coli
These are some of the tools available to get around some of the common issues with low protein yield.
Coming up in this series we will look more closely at handling issues with mis-folding proteins, as well as how to employ cell-free expression systems.
Until then, if you would like some help figuring out how to best express your troublesome proteins, send us an email at info@bionordika.se and we’ll be happy to help out!
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