Optimizing Protein Expression with IPTG Induction
by Simon Currie
The final concentration of IPTG used for induction varies from 0.1 to 1.0 mM, with 0.5 or 1.0 mM most frequently used. For proteins with high solubility 0.5 mM or 1.0 mM IPTG enables high protein expression. However, minimizing IPTG may work best for proteins with low solubility.
Isopropyl b-D-1-thiogalactopyranoside (IPTG) induces recombinant protein expression in bacteria. But have you ever used your lab’s standard IPTG induction protocol and been disappointed with your protein’s meager expression?
If low protein expression has you feeling frustrated, then this is the article for you! We’ll discuss variations of standard IPTG protocols and walk through optimizing IPTG induction for difficult-to-express proteins.
The final concentration of IPTG used for induction varies from 0.1 to 1.0 mM, with 0.5 or 1.0 mM most frequently used. For proteins with high solubility 0.5 mM or 1.0 mM IPTG enables high protein expression. However, minimizing IPTG may work best for proteins with low solubility.
For some proteins, exactly how you optimize induction will differ depending on whether you’re trying to maximize the amount of soluble protein or instead trying to drive your protein into insoluble inclusion bodies. Let’s dive in.
Article Table of Contents
When and when not to optimize your protocol
About the two standard induction protocols
Optimizing insoluble protein expression
IPTG induction
IPTG induction is a very common way to initiate protein expression, but how exactly does it work? Well, at the transcriptional level, there is a protein called the lac repressor, which is aptly named because it binds to a DNA sequence called the lac operator and blocks transcription (Figure 1).
Figure 1. When the lac repressor is bound to the lac operator, it blocks RNA polymerase from transcribing downstream genes.
Lac is short for lactose, and in their native biological context the lac repressor and lac operon serve as a “sensor” so that genes involved in the lactose metabolism are only expressed when lactose is present (Monod et al, 1965; Simas et al, 2023).
During lactose abundance, the derived metabolite allolactose binds to the lac repressor and reduces the protein’s affinity for DNA, effectively kicking the lac repressor off of DNA and allowing transcription of the downstream gene to proceed (Figure 2) (Razo-Mejia et al, 2018).
Figure 2. Allolactose or IPTG binding to the lac repressor weakens its binding to DNA, allowing RNA polymerase to proceed through and transcribe the downstream gene(s).
IPTG is a synthetic analog of allolactose, meaning IPTG does the same thing: it evicts the lac repressor from binding to the lac operon, thereby activating transcription.
The advantage of IPTG is that it is not metabolized by the cell, unlike allolactose. So, during the hours that you’re inducing your protein expression, the IPTG concentration will remain constant and will not decrease due to being converted by cellular enzymes into other things.
When and when not to optimize your protocol
In a few sections we’ll discuss how to optimize IPTG induction for a single protein. Some will tell you that you need to optimize the expression protocols for every single protein that you work with.
In my opinion, that might be a little more effort than necessary. Standard induction protocols are great because they often work well enough to supply you with sufficient protein for your required experiments. And you save the time you would have spent optimizing every single protein that you work with.
However, if at least one of the following applies to your protein expression, then yes, it is probably worth optimizing its expression:
· The standard protocol doesn’t work very well for your protein of interest
· You’re going to be making a ton of this protein over and over again
If neither of the above applies, or it’s your first time working with this protein and you’re not sure how much protein you’ll need, then one of the following two standard expression protocols are what most people use.
· 1 mM IPTG, 2-4 hours induction @ 37 or 30 °C.
· 0.5 mM IPTG, overnight @ 18 °C
About the two standard induction protocols
The two standard protocols vary in multiple parameters (IPTG concentration, time, and temperature), so let’s discuss the logic behind these conditions. First, typically the higher the concentration of IPTG, the higher the total protein expression will be.
The first protocol uses 1 mM IPTG, but only for 2-4 hours at 37 or 30 °C. This is the hot and fast protocol. You’ll produce so much of your protein in the first few hours that it exhausts the cells’ production machinery. So, you could keep going for longer, but you’re probably not going to get any more protein out of it.
This expression condition is good when you know your protein is very soluble, and also if your protein is very insoluble and you’re trying to force it into inclusion bodies.
The second protocol gradually expresses the protein both by using less IPTG (0.5 mM) and by expressing the protein at 18 °C. Generally, this will help your protein express into the soluble fraction and prevent your protein from going into inclusion bodies. Since it is expressing at a lower level, you usually want to let the expression go for longer. Somewhere between 6 to 12 hours is optimal for most proteins, but that would be in the middle of the night, so most scientists just let it go overnight and harvest the cells first thing the next morning.
Optimizing insoluble protein expression
When your protein expresses insolubly, then it will accumulate into inclusion bodies. Functionally, this means that after you lyse the bacterial cells and centrifuge them, the proteins will pellet with cellular debris like membranes, etc. We discuss inclusion bodies and how you can recover your protein from them in this article.
When your protein is expressing insolubly, you have two options. You can optimize your protein expression construct or the expression conditions to try to maximize the soluble fraction and minimize the insoluble fraction of your protein. Or, you can lean into the insoluble expression and try to force as much of the protein as possible into inclusion bodies.
If you’re trying to promote your protein’s expression into inclusion bodies, then you’ll want to go with the hot and fast standard protocol. Then you can isolate the inclusion bodies and refold your protein out of them.
Alternatively, if you’re trying to maximize the soluble expression, then you’ll want to go with the low and slow protocol as a starting point, and maybe go even lower from there. Meaning, you could lower the IPTG concentration even further to promote more soluble proteins (Figure 3). Also, there are specialized bacteria that express proteins well at even lower temperatures, so that is another option you could try if your protein is particularly difficult (Ferrer et al, 2003).
Figure 3. Left, very soluble proteins typically have higher expression with the hot and fast induction (pink line), whereas proteins with a broader range of solubilities will have more soluble expression with the low and slow induction (blue line). Right, hot and fast induction is better for driving proteins into inclusion bodies, but this will only work for proteins with poor solubility.
In the next section, we’ll discuss how to optimize IPTG induction. However, some proteins won’t express well into the soluble fraction even under optimized conditions. If this is the case for your protein, there are a few things to consider:
Protein design considerations:
- Did you design your protein correctly?
- Did you accidentally truncate the protein in the middle of a domain?
- Are there other protein segments you could try that would work for your experimental needs?
Solubility tag considerations:
- Have you tried using a solubility tag?
- GST-, MBP-, and SUMO-tags can assist proteins expressing into the soluble fraction in bacteria.
Expression system considerations:
- Have you tried another expression system?
- There are lots of bacterial variants you could try, but also, not every protein will express in bacteria.
- You may need to try insect or mammalian cell expression instead.
Optimizing IPTG induction
You’ve probably gathered that there are a lot of variables that can be adjusted while optimizing protein expression. The two that I would start experimenting with first are IPTG concentration and temperature.
Using small scale ferments (~5-10 mL), you can quickly scan through these parameters to see what works best for your protein (Figure 4).
Figure 4. When optimizing induction conditions, you can analyze small-scale ferments with different temperature and IPTG concentrations. Each tube represents a small expression with distinct combinations of IPTG and temperature.
The total protein yield won’t tell you how much of your protein is soluble and how much is insoluble. So, when doing these small-scale test ferments, it will be worth lysing the cells and centrifuging the lysate to separate the soluble and insoluble fractions. Then you can run samples of each on an SDS-PAGE gel to analyze them. It’s useful to include a control sample where you didn’t induce protein expression to help differentiate your protein of interest from host proteins (Figure 5).
Figure 5. Examining a single induction condition on an SDS-PAGE gel. This protein of interest expresses more in the soluble fraction (sol) than inclusion bodies (ins).
These results will help direct your large-scale ferments and protein purifications.
If most of your protein is in the soluble fraction, then use the conditions that give you the most protein (Figure 6).
Figure 6. Most of the protein of interest is in the soluble fraction under all induction conditions, so select the condition that gives the most soluble protein, 1 mM IPTG in this case, and use that do induce your scaled-up ferment.
If your protein predominantly expresses into the insoluble fraction, either pick the conditions that give you the most insoluble protein and try to purify and refold your protein from inclusion bodies (Figure 7), or revisit our tips above to try to design a new expression construct that will express soluble protein.
Figure 7. Most of the protein of interest is in inclusion bodies under all induction conditions. Either select the condition that gives the most insoluble protein, 1 mM ITPG in this case, and use that induction for your scaled-up ferment, or try additional conditions or expression constructs to coax your protein into the soluble fraction.
If your protein expresses both into the soluble and insoluble fractions depending on the conditions (Figure 8), then you have choices about how you want to purify your protein. Usually, it is much less of a hassle to purify soluble proteins. However, there can be benefits to going the insoluble route in certain cases, such as increased protein purity (Currie et al., 2017).
Figure 8. This protein of interest expresses both into the soluble and insoluble fractions, at different proportions depending on the expression conditions. If you want to focus on the soluble protein, then 0.25 mM IPTG is the best condition tested. But 1 mM IPTG gives the most protein overall, albeit mostly in inclusion bodies.
That’s how to optimize IPTG induction for your protein of interest. Remember, in many cases a standard induction protocol will work fine and you won’t even need to do these optimization tests. However, if your protein failed with a standard protocol or if you’ll be purifying a lot of the same protein over and over again, then it will definitely be worth your time to define the optimal expression conditions.
At GoldBio we have a lot of reliable and affordable reagents that can assist you with your protein induction and purification needs, as well as resources to help guide you on your way. Check some of those out below!
References
Currie, S. L., Lau, D. K. W., Doane, J. J., Whitby, F. G., Okon, M., McIntosh, L. P., & Graves, B. J. (2017). Structured and disordered regions cooperatively mediate DNA-binding autoinhibition of ETS factors ETV1, ETV4 and ETV5. Nucleic acids research, 45(5), 2223–2241. https://doi.org/10.1093/nar/gkx068
Ferrer, M., Chernikova, T. N., Yakimov, M. M., Golyshin, P. N., & Timmis, K. N. (2003). Chaperonins govern growth of Escherichia coli at low temperatures. Nature biotechnology, 21(11), 1266–1267. https://doi.org/10.1038/nbt1103-1266
MONOD, J., WYMAN, J., & CHANGEUX, J. P. (1965). ON THE NATURE OF ALLOSTERIC TRANSITIONS: A PLAUSIBLE MODEL. Journal of molecular biology, 12, 88–118. https://doi.org/10.1016/s0022-2836(65)80285-6
Razo-Mejia, M., Barnes, S. L., Belliveau, N. M., Chure, G., Einav, T., Lewis, M., & Phillips, R. (2018). Tuning Transcriptional Regulation through Signaling: A Predictive Theory of Allosteric Induction. Cell systems, 6(4), 456–469.e10. https://doi.org/10.1016/j.cels.2018.02.004
Simas, R. G., Pessoa Junior, A., & Long, P. F. (2023). Mechanistic aspects of IPTG (isopropylthio-β-galactoside) transport across the cytoplasmic membrane of Escherichia coli-a rate limiting step in the induction of recombinant protein expression. Journal of industrial microbiology & biotechnology, 50(1), kuad034. https://doi.org/10.1093/jimb/kuad034
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