The interaction between the small molecule biotin and the protein streptavidin is incredibly strong and has been co-opted for numerous biotechnological purposes. For example, by biotinylating proteins, or covalently conjugating biotin to a protein, you can use the biotin-streptavidin interaction for detecting, tracking, and isolating your protein of interest as well as its interacting partners.

There are several ways to biotinylate a protein. Perhaps the most convenient way is to site-specifically biotinylate an avi-tagged form of your protein while it is being expressed. 

An avi-tag fusion of your protein of interest may be biotinylated while it’s being expressed in E. coli or other expression systems. Co-expressing the biotin ligase BirA and supplementing the medium with biotin ensures efficient and robust biotinylation, specifically on the avi-tag.

In this article, we’ll briefly discuss what an avi-tag is, how to biotinylate an avi-tagged protein during expression, and some of the uses for biotinylated proteins.  

Table of Contents:

What’s an avi-tag?

How to biotinylate a protein during expression

Why biotinylate a protein during expression?

Other options for biotinylating proteins

Considerations for biotinylating proteins during expression

Experiments with biotinylated proteins

References

 

What’s an avi-tag?

An avi-tag is a short affinity tag that the biotin ligase BirA binds to and then conjugates biotin to a specific lysine residue within the avi-tag (Figure 1).

Figure 1. BirA catalyzes biotinylation of the lysine residue in an avi-tag.

 

Compared to other methods of biotinylating proteins, using an avi-tag results in specific biotinylation on one lysine residue within the tag, which we’ll discuss more in the section below about other options for biotinylating proteins.

 

How to biotinylate a protein during expression

There are four key ingredients for biotinylating a protein during cellular expression:

1. A protein-expression system such as bacterial, insect, or mammalian cells
2. A plasmid that expresses an avi-tagged form of your protein of interest
3. A plasmid that expresses the biotin ligase BirA
4. Biotin

Following standard protocols, you’ll co-express your avi-tagged protein of interest and BirA at the same time in your protein-expression system of choice. You’ll want to supplement the media with extra biotin during protein expression to ensure robust biotinylation (Figure 2).

Figure 2. Coexpression of BirA (orange) and your protein of interest (POI, green) from separate plasmids leads to biotinylation of the avi-tagged POI inside the E. coli cell (dark blue).

Conceptually, biotinylating your avi-tagged protein is quite simple.

 

Why biotinylate a protein during expression?

There are other ways to biotinylate a protein besides during cellular expression. We will discuss those other methods briefly in the next section, and in more depth in this article.

The reason that I usually first attempt biotinylation during protein expression is because if it works, it is the easiest method. Biotinylating during protein expression allows the cells to do the hard work and biochemistry of biotinylation, and saves you an extra couple of steps and a few hours of hands-on time during your purification scheme.

Unfortunately, biotinylation during protein expression doesn’t work for every protein. It’s not always exactly clear why some proteins struggle with in vivo biotinylation, but the common phenotype is that you’ll get really poor expression of your protein of interest (Figure 3).

Figure 3. Hypothetical SDS PAGE gels showing protein expression with (+) and without (-) biotinylation. Left, similar expression, or even a modest decrease in expression means that cellular biotinylation worked well. Right, if you have a major decrease in expression for your protein of interest, then try biotinylating the purified protein after you’ve already expressed it.

Two hypotheses for the poor expression are that co-expressing your protein of interest and BirA causes extra metabolic stress on the cells, or that the biotinylated protein has poor solubility compared to the unbiotinylated or non avi-tagged form (Li and Sousa, 2015).

So, if you try co-expression with BirA first and you get very little protein, then you should try expressing the avi-tagged protein of interest by itself, then biotinylating the purified protein as part of your purification protocol to see if that enhances your final protein yield.

 

Other options for biotinylating proteins

Instead of biotinylating proteins while they’re being expressed, you can biotinylate purified proteins using chemical or enzymatic methods. We discuss those methods in more detail in this article.

First of all, your protein needs to have an avi-tag if you want to biotinylate it enzymatically with BirA, regardless of whether that is during co-expression or biochemically with purified proteins. So, if you have an avi-tagged protein you have options here, but if you don’t have an avi-tag on your protein then you’ll either be chemically biotinylating your purified protein, or back to the cloning stage to generate an expression plasmid with an avi-tagged form of your protein.

If your avi-tagged expression vector doesn’t express your protein very well when co-expressing with BirA, you should definitely try expressing your protein by itself, and then enzymatically biotinylate the avi-tagged protein with recombinant BirA during your purification protocol.

 

Considerations for biotinylating proteins during expression

There are a couple of decisions to consider when biotinylating avi-tagged proteins during expression:

·         What cellular expression system are you using?

·         What plasmids are you using for co-expression?

 

Choosing a cellular expression system

Co-expression with BirA has been successful in many common protein expression systems, including E. coli, insect cells, and mammalian cells (Erdmann et al, 2010; Ioannou et al, 2018; Li & Sousa, 2012).

If your protein has been successfully expressed in any of these expression systems, then I would try that same expression system first. If you are new to working with this protein, make sure to scour the literature to see what other scientists have done when working with your protein, or a similar, evolutionarily-related protein because that can give you hints as which system to try first.

 

Designing plasmids for co-expression

Knowing which protein expression system you’re using will help you choose which kind of plasmids you should use for co-expression.

Above, I set up co-expression as always using two separate plasmids, one for BirA, and one for your avi-tagged protein of interest (Figure 2). Alternatively, you could design one plasmid that has two multiple cloning sites, one for BirA and one for your avi-tagged protein of interest (Figure 4).

There’s no absolute best choice here. Using two expression plasmids makes it easier to plug and play by combining your BirA plasmid with any different avi-tagged protein plasmid that you want to biotinylate. However, when using two different plasmids it is important to make sure that they have different antibiotic resistances (when your cell expression system uses antibiotic resistance). For example, one plasmid could have kanamycin resistance and the other could have ampicillin resistance (Figure 5).

Figure 5. When expressing two separate plasmids, they need to have resistance to different antibiotics, such as ampicillin (left) and kanamycin (right).

 

Also, if using two separate plasmids it is important for each plasmid to have different and compatible origins of replication (ORIs). If they have the same origin of replication, the plasmids will compete for the endogenous DNA replication machinery, which will ultimately limit your protein’s expression (Figure 6) (Morgan, 2020).

Figure 6. Plasmids with different and compatible origins of replication (ORIs) use different DNA replication proteins and can therefore both be efficiently replicated (left). Plasmids with similar ORIs compete for the same DNA replication machinery, which will limit plasmid propagation and ultimately protein expression (right).

 

By the way, if you want to review what multiple cloning sites and origins of replication are, this article that describes 5 key elements of plasmids is a great resource for that.

Using a single plasmid allows you to skip multiple antibiotics (which slows cell growth) and you won’t have to worry about whether the origins of replication are compatible.

However, if you’re going to be biotinylating a lot of different proteins during their cellular expression, you will have to re-clone each one into the single co-expression plasmid alongside BirA. If you think that will be your case, then it may be worth it to have a single plasmid expressing BirA, and a separate compatible plasmid that expresses each of your different proteins of interest. 

 

Experiments with biotinylated proteins

Biotinylating a protein is really helpful for detecting and quantifying that protein, as well as isolating that protein and any interacting partners.

There are many powerful experiments that leverage the biotin-streptavidin interaction including: Western blots, enzyme linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and streptavidin pull-downs, microscopy and imaging including immunohistochemistry and immunofluorescence, and more.

We discuss these assays in this article in the context of biotinylated antibodies. However, keep in mind that most of these experiments are powerful for all kinds of proteins, not just antibodies. 

 

By now you can probably appreciate just how easy it is to biotinylate proteins while they’re being expressed. Below we have lots of great resources to help you learn more about how and why to biotinylate proteins. Additionally, GoldBio has many useful and reliable reagents for biotinylating proteins and also conducting experiments with these versatile reagents. Check out all of those links below, and you’ll be well on your way to becoming a biotinylation boss!

 

 

Tags

biotin Biotin-NH2 Biotin-SH Simon Currie streptavidin-biotin

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