The protein streptavidin clamps onto the small molecule biotin in one of the tightest natural interactions ever discovered. Over the years, the strength of this interaction has been used to develop clever protein biotechnology tools by biotinylating proteins which is when biotin is conjugated onto a protein.

Proteins are biotinylated using either chemical reagents which add biotin to certain types of residues throughout the protein, or enzymatically to a specific amino acid. Adding biotin enables researchers to track, quantify, and isolate the protein of interest and its molecular partners.

In this article, we’ll discuss these two main methods for protein biotinylation and compare and contrast their strengths and drawbacks. We’ll also briefly cover the kinds of experiments that protein biotinylation enables.

Table of Contents

Methods for biotinylating proteins

Chemical labeling throughout the protein

Enzymatic labeling of Avi-tags

Experiments with biotinylated proteins

Tracking and visualizing biotinylated proteins

Isolating biotinylated proteins and their interaction partners

Proximity labeling

References

 

What are the methods for biotinylating proteins?

There are two main ways to biotinylate proteins:

• Chemical biotinylation
• Enzymatic biotinylation

There are tradeoffs for each of these methods. Chemical biotinylation is usually the faster approach, but typically adds biotin molecules redundantly, meaning more than one biotin is added per protein molecule, and these labels are added throughout the protein.

Enzymatic biotinylation, on the other hand, adds one biotin to an Avi-tag, but will require you to re-clone your protein expression plasmid if your protein doesn’t already have an Avi-tag.

 

Chemical labeling throughout the protein

The “chemical” method adds a biotin to certain types of amino acids, usually cysteine or lysine depending on which kind of chemical you’re using.

Maleimide-biotin chemicals react with the thiol on a cysteine residues side chain to form a covalent bond between the protein and biotin. Analogously, N-Hydroxysuccinimide (NHS)-biotin chemicals react with the amide on lysine’s side chain to attach biotin to the protein (Figure 1). 

Figure 1. Cysteine residues conjugate to maleimide biotin (top) whereas lysine residues do so with NHS biotin (bottom).

Cysteines are one of the least abundant residues in most proteins. If your protein only has one cysteine, then that is the only location that it will be biotinylated with maleimide reagents. So, in that sense you get kind of lucky based on your protein’s natural composition. Similarly, if your protein has no cysteines then you can mutate a specific residue of your choosing to cysteine, and that will be the only site for maleimide biotinylation.

However, if your protein has more than one cysteine residue then you will get redundant biotinylation at any surface-exposed cysteine (Figure 2).

Why does this matter? Well, ideally you want the biotinylation of your protein to not interfere with its function. If the biotin is conjugated to an active site of an enzyme, or at the interface with an important interaction, then biotinylation may disrupt that protein’s function.

Figure 2. Chemical method usually redundantly biotinylates a protein (left), whereas the enzymatic method specifically biotinylates a protein on its Avi-tag (right

 

So, in cases where there is more than one cysteine present in a protein, researchers will often mutate out cysteine residues until there is only one left. Alternatively, they label the wild-type protein and use some kind of functional of physical assay after the biotinylation reaction to ensure that the biotinylated protein retains activity.

Lysine residues are more abundant than cysteine, so typically you will get redundant biotinylation when using NHS Esters to label lysine residues. We cover NHS labeling of lysine residues in detail in this article, so check that out if you want more information. Again, this is where it would be important to check the protein’s activity and determine if biotinylating lysine residues disrupt its function.

If you know that your protein has a crucial cysteine or lysine residue, then you can either use the other chemical biotinylating reagent, or you can go with enzymatic biotinylation instead.

 

Enzymatic labeling of Avi-tags

Instead of redundantly biotinylating throughout your protein with chemical reagents, an alternative is to add an Avi-tag onto your protein and specifically biotinylate only a single lysine residue within the Avi-tag (Figure 2). BirA recognizes and binds to the Avi-tag sequence, which then leads to it biotinylating only the lysine within the Avi-tag, rather than on lysines throughout the protein like in the chemical method.

An Avi-tag is a specific peptide sequence that is biotinylated by an enzyme called BirA (Figure 3). You can biotinylate your Avi-tagged protein either by coexpressing BirA with your protein of interest and adding biotin to the media, or by incubating BirA and biotin with your partially purified protein (Fairhead & Howarth, 2015).

Figure 3. BirA catalyzes biotinylation of the lysine residue in the Avi-tag.

 

The downside of this approach is that it only works for Avi-tagged proteins. So, if your protein currently lacks an Avi-tag then you’re back to the cloning stage. However, with an Avi-tag you know that your protein has been biotinylated once and you know exactly where that biotin is located. For these reasons, Avi-tags less frequently disrupt a protein’s function as compared to chemical biotinylation.  

 

Experiments with biotinylated proteins

Biotinylating a protein is really helpful for detecting and quantifying that protein, as well as measuring interactions with other molecules.

Tracking and visualizing biotinylated proteins

Biotinylation is useful for visualizing a protein. For example, you could track where a protein is in the cell, or you could use that visualization to quantitate how much of the protein is present. These applications utilize something called chemical luminescence where biotin binds to a streptavidin molecule that is conjugated to an enzyme called horseradish peroxidase (HRP) that produces light (Figure 4). So, in this setup you’re indirectly measuring your protein of interest through this chemiluminescent signal.

Figure 4. HRP (red) fused to streptavidin (blue) creates a chemiluminescent signal that is used to track your target biotinylated proteins (green).

 

Isolating biotinylated proteins and their interaction partners

Biotin is also a useful handle to “grab” your protein and isolate it and any interaction partners.

For example, you can use streptavidin agarose beads to isolate your biotinylated protein. Prior to grabbing your biotinylated protein, you can use it for an experiment like incubating it with another protein that you think it may interact with, or incubating it with cell lysate to find new proteins that it interacts with (Figure 5).

Figure 5. Streptavidin beads (dark blue circle) immobilize biotinylated target proteins and any interacting proteins they bind to.

 

Proximity labeling

There are proximity labeling experiments that biotinylate nearby proteins by fusing BirA to a protein of interest (Figure 6). These experiments are usually conducted in cell lysate, or more commonly in intact cells by genetically expressing the BirA-Protein of Interest fusion. This kind of experiment is known by names such as BioID and TurboID, and were developed in Dr. Alice Ting’s lab (Qin et al, 2021).  

Figure 6. BioID and TurboID use BirA (orange) fused to a target protein (green) to biotinylate and identify interacting proteins (pink). The high local concentration of the interacting protein drives BirA mediated biotinylation on its lysine residues.

 

BioID and TurboID use BirA to label nearby proteins on lysine residues throughout those proteins, which is a little bit different than what we’ve talked about for the in vitro use of BirA for specifically labeling lysine in an Avi-tag. BirA is enzymatically biotinylating nearby proteins, but it is now doing so on lysine residues throughout the protein, which is conceptually more like the chemical mechanism that we discussed above. This difference essentially comes down to concentration. In proximity labeling experiments, BirA has a high local concentration because the protein of interest that it is fused to is interacting with other proteins that BirA is biotinylating.

In the in vitro context if you used a really high concentration of BirA you could also biotinylate lysine residues besides the one in the Avi-tag. Typically, it is recommended to use one BirA molecule per 100-200 molecules of Avi-tagged protein. So, you would need to go much higher than this ratio to have BirA biotinylate lysines outside of the Avi-tag. 

 

So, that’s all about how to biotinylate proteins to enable a variety of powerful experiments. If you need to biotinylate your protein of interest, we have lots of reliable and affordable reagents to help you on your way, so check those out below. Additionally, we have a lot of other resources regarding biotinylated proteins and labeling proteins with other molecules such as fluorophores which you can also find below if you want to learn more.

 

 

References

Fairhead, M., & Howarth, M. (2015). Site-specific biotinylation of purified proteins using BirA. Methods in molecular biology (Clifton, N.J.), 1266, 171–184. https://doi.org/10.1007/978-1-4939-2272-7_12

Qin, W., Cho, K. F., Cavanagh, P. E., & Ting, A. Y. (2021). Deciphering molecular interactions by proximity labeling. Nature methods, 18(2), 133–143. https://doi.org/10.1038/s41592-020-01010-5

 

Tags

biotin Biotin Labeling Kit Biotin-NH2 Biotin-SH protein expression Protein Induction protein purification Simon Currie streptavidin-biotin

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