Last time on Troubleshooting, we covered Tagged Protein Purification, but what happens if your protein of interest isn’t being expressed in the first place!
Induction of a bacterial protein should be pretty straightforward. You add your inducer to your culture, and immediately your cells are transformed into magical protein production powerhouses, churning out copy after copy of your protein of interest.
Unfortunately sometimes instead of being production powerhouses, your cells just lazily make your protein here or there, or maybe don’t pick up the job at all!
Here at GoldBio, we get a lot of questions about bacterial protein induction, and in this edition of Troubleshooting we’ll address some of the most common issues so we can get those cells working in high gear!
Common Factors Affecting Protein Expression
There are main factors to consider when optimizing induction conditions: Vector, Host Strain, and Growth Conditions. These three factors have the biggest impact on the expression of your protein of interest.
First on the list of consideration is the vector you’re using to express your protein. There are countless bacterial expression vectors that are commercially available, each with their own positive and negative aspects. There are vectors designed to express toxic proteins, or proteins that contain long stretches of repeating sequence.
But no matter what vector you decide to use, the first thing to do is make sure that after cloning, your protein of interest is still in frame. It’s recommended that before you run any expression studies that you first have your cloned plasmid (or a few different clones) sequenced. This will show if the sequence you inserted into the expression vector is still correct and is still in frame.
This is especially important if the construct contains any PCR fragments or if the construct was made via Gibson Assembly or other enzymatic method. If there are any point mutations or the sequence gets out of frame by even a few bases it can have dramatic effects on the protein you are trying to express.
Another thing to check before expressing is if your protein sequence contains long stretches of rare codons. This can cause the protein that is expressed to be truncated or non-functional. A few rare codons spread around the protein are OK in most cases, but if there are a number of rare codons in a row, then it can have a big effect.
There are a number of online tools to help analyze this data, and if there are a detrimental number of rare codons, you can either try to modify them by point mutation or use an expression host that will boost the levels of the necessary tRNA for correct translation.
The third sequence related step you can take to optimize your protein production is to make sure there isn’t a high GC concentration at the 5’ end of your protein. This could potentially cause problems with the mRNA’s stability, and could prevent it from being translated correctly, which would also lead to truncated or non-functional proteins. If your sequence is GC heavy at this end, you can try to make a few silent mutations to break up long stretches to try and help stability.
2. Host Strain
After your plasmid is sequence verified, the next factor is the bacterial host you’re using. There are almost as many hosts as there are expression vectors, with certain hosts excelling at producing different types of proteins.
For example if you have a toxic protein, or a protein that could potentially cause genomic rearrangement, you will want a vector that gives you very tight control over the induction of your protein.
There can be “leaky” expression (i.e. expression of your protein without the addition of your inducer) that can potentially have adverse effects on the cells growth or even prevent your cells from over-expressing your protein in the first place. If you’re utilizing the T7 polymerase system, then look for a host containing the pLysS plasmid, as this will code for T7 lysozyme, which will suppress the T7 polymerase and can greatly reduce the level of background expression.
If as stated before you have a protein that contains a large number of rare codons, then look for a host with the genes for the necessary tRNA’s already present, which should allow your protein to express correctly. Sometimes simply changing hosts can have a dramatic effect on the amount of protein produced and the stability of the protein that is made, so if one host isn’t giving you the results you need, then feel free to switch your host up.
3. Growth Conditions
The third and final factor to consider when expressing a protein is growth conditions. When first starting out with your protein induction it is very important to run an expression time course, where you take a fresh colony from a streaked plate, and grow the culture to stationary phase.
Next, dilute the overnight culture 1/100 and grow to mid log phase, then add the inducer and induce your protein for a number of hours, taking 1mL samples every hour or so.
Once these samples are lysed, you can run an SDS-PAGE gel to determine your protein production levels. You might get great induction the first time, or you may have to tweak your conditions in order to get really good expression levels.
Other factors that may need to be controlled for are the bacterial growth rate (determined by taking OD measurements during the induction process), and the temperature during induction. Some constructs will express perfectly fine at 37°C, while others need to be bumped down to 30°C to induce correctly.
The concentration of your inducer too will have an effect, as many inducers (such as IPTG) can be toxic to the cells that they are inducing.
Using freshly made inducer is good step to making sure you always have consistent results. Only through experimentation can you determine what will be best for your construct, and give you the most robust expression levels.
Optimizing your protein induction can be a very labor intensive and time consuming step, but if all the precautions are taken, and if the experimentation is done with great care, your cells will be pumping out your protein like the little factories we desire. And you’ll be ready for the next big step in your research! As always, if you have any questions you can always call or email us here at GoldBio, and there are a myriad of resources online full of people ready to help.
Category Code: 79105 79102 88253 79103 88231 79108 79107