In the first part of this series, we covered the basic steps for developing an enzyme assay. Now that we got the basics out of the way, let’s share a few ‘pro tips’ for developing enzyme assays for trickier scenarios. 

Tip #1: Employing Enzyme Teamwork

Sometimes when you are developing an enzyme assay, you may be faced with a challenge: neither the substrate nor the product of the reaction can be easily measured, nor does it require a cofactor that could be used as a proxy. How can you study the kinetics of the reaction you cannot measure? 

One way to get around a situation like this is by using enzyme coupling: adding a second enzyme which utilizes the product of the first reaction to make something that can be easily measured. This is a trick we call ‘enzyme teamwork’. You can use a commercially available enzyme to create an enzyme coupling system or synthesize one in the lab yourself. 

One thing to keep in mind when using enzyme coupling is that you want the coupling enzyme to be very efficient (or abundant) to ensure that the reaction proceeds forward at a high rate. Otherwise, it will become the rate-limiting step, which means you will not be able to capture the kinetics of the enzyme you are interested in. 

Alternatively, enzyme teamwork can be utilized on the front end of the reaction. In cases when the substrate you need to use is not commercially available or easy to synthesize in-house, you can make it directly in the plate via another enzyme. You will want to identify a chemical precursor and an enzyme that can convert it into your substrate of interest. The same advice about the efficiency of the precursor enzyme applies here to ensure that the substrate production does not become a bottleneck. 

Tip #2: Using In Vivo Assays

Developing an enzyme assay does not necessarily mean that you have to break the cell and purify the protein of interest. You can create an in vivo enzymatic assay to measure the reaction as it is happening in the cell. This can be an incredibly powerful approach— for instance, if you are working with an enzyme that is membrane-bound and cannot be isolated.

Many people think that developing high-throughput in vivo assays is hard and not worth the trouble. But it gives you the opportunity to measure the enzyme activity in the most perfect environment: inside the cell. In fact, some of the enzyme variants that work very well in in vitro screenings do not perform as well in living cells. An in vivo enzyme assay can help avoid this problem. This approach can be applied not only to enzymes but to other proteins, such as transporter proteins, as well (see example below).

Example: An in vivo assay for measuring the activity of a transporter protein. 

If you are measuring something that relies on the cell staying alive and functional—such as active transport of a cellular compound—you cannot use in vitro assays that require cell lysis. One of the proteins Jinel worked on at a previous company was exactly that: a transporter that could only function in living cells. However, measuring its function in vivo was impractical in this specific case.

The way she got around this restriction was by designing a clever two-step hybrid assay. She grew the cells that expressed the transporter and produced the chemical that needed to be exported. The reasoning was: if the transporter is doing its job, it should transport the chemical produced inside the cell to the outside while the cells are alive. However, once the product was transported outside of the cell, she did not have to keep them alive anymore.

To measure the transporter activity, she spun the cells down and used the supernatant as a substrate for a second enzyme that converted it into a product she could easily measure. The second in vitro enzymatic assay served as a proxy for determining the in vivo activity of the transporter she was interested in.

Tip #3: Improving Protein Expression

One of the important steps in developing a biochemical assay is optimizing the protein expression. Solubility is a common problem encountered during protein expression and purification, especially for membrane proteins such as transporters. When you break apart a cell, these proteins do not stay in solution but precipitate with the rest of the cellular debris. In this case, you can try developing an in vivo assay, as we suggested in Tip #2. 

Expression can also be a problem for some toxic proteins. Either due to their innate toxicity or because they generate a toxic product (or byproduct), expressing those proteins at high concentrations is toxic to the cell. Additionally, if you are trying to express a protein from a different organism in a heterologous expression system, it can cause the protein to misfold and, as a consequence, become insoluble. 

To help improve protein expression, you could try lowering the temperature the cells are grown at or use ‘protein tags’ to help with expression. You can also use enzyme teamwork here: some proteins fold and stay in solution so well that if you couple them with your enzyme/protein of interest they will help improve its expression and solubility as well.

However, sometimes none of these tricks work. In this case, you may need to go back to the drawing board to make certain mutations in the enzyme that reduce its toxicity or help improve its folding and solubility. Finally, if you have exhausted all possibilities, and have to accept that the protein you are working with expresses at a very low concentration, you can just use a more sensitive fluorescence- or luminescence-based assay or a cascade of enzymes to amplify the signal. 

Tip #4: Purifying and Quantifying the Enzyme

Isolating, purifying and quantifying the enzyme is important when you are first developing an assay. A pure enzyme sample is required for determining the enzyme metrics like kcat and km. It also ensures confidence in the assay readout, since conducting the assay in cell lysate could add noise. Once you have validated the assay, however, you will most likely be able to conduct it in cell lysate, without needing to purify the enzyme, especially if it shows high expression and measurable activity. 

To get a high enough expression that enables doing the assay in cell lysates, you can express your enzyme of interest on a plasmid. Utilizing constitutive expression or fine-tuning the enzyme induction levels can help you achieve high expression and purity. Including an empty vector control is a good way to ensure that your signal comes from the enzyme and not the host. 

If you need to quantify the enzyme, you also don’t necessarily need to purify it. You can add a green fluorescent protein (GFP) tag to quantify the protein in lysate. Additionally, if your enzyme is expressed on a plasmid, you may not need to do that at all. With plasmid-based expression, you have enough protein to estimate its expression level by simply running a sample of the cell extract on an SDS-PAGE gel. This should be a routine practice, in combination with OD normalization, to ensure that the variance you see in the assay is not from protein expression or cell growth differences.

Final thoughts

We hope that these posts have given you confidence in being able to develop basic enzyme assays and even tackle some of the more tricky ones. 

Every enzyme is unique—and so is every assay. Developing a new assay is an exciting challenge and an opportunity to get creative in the lab. Often, the hardest thing is just the first step: deciding which direction you want to take in your assay design process. There are so many ways to engineer an enzyme that trying to figure out exactly which threads to pull can lead you on a wild goose chase. So, just start somewhere and see where the creative journey takes you.