How to Use Proteases to Purposefully Digest Proteins

In this article I will not talk about ‘wild’ proteases, which destroy cellular proteins in your lysates like wolves destroy sheep. Instead, I’ll be talking about the shepherd dog proteases—purified, tame and useful to digest proteins your research.

In Protein Research and Crystallization

Several programs can predict your protein domains. However, we wet biologists know that there is always a gap between a prediction and reality. The proof is in the pudding or, rather, in the lab. If your protein is impossible to clone and/or crystallize, you need to determine protein domains experimentally.

And one of the ways to do so is by using proteolytic enzymes to digest proteins.¹ Using proteases to probe you protein structure is a bit like using frequently cutting restriction enzymes on DNA for a partial digest.

In a nutshell, use a proteolytic enzyme in vitro to partially digest your protein. The interdomain loops are more susceptible to enzymatic cleavage than structured domains, and the protease should release individual domains.  Vary the enzyme concentration and the time of the proteolysis to get various stages of protein digestion. After partial proteolysis, run the digest on a gel visualize the products as protein bands. You can assess the sequence of the bands by NMR or LS-MS, which will allow you to determine the real boundaries of your domains.

See this sample protocol and its references for more details.

In Mass Spectrometry

In LS-MS, you don’t determine the tertiary structure of your proteins, but their primary structure and changes to it. For example, you can check your protein’s posttranslational modifications or presence of splicing variants in different tissues.

For this, use proteases to make your protein able to “fly” by reducing the size of the fragments. Digest proteins in solution or in the gel. Then, a scientific officer will fly your molecules for you and provide the result.

Common Proteolytic Enzymes to Digest Proteins

Proteases may be less popular than enzymes that digest nucleic acids, but proteases are still quite common. You just need to know your predicted protein sequence and plan accordingly.

Here are some of the most-used ones:

• Trypsin – the most commonly used proteolytic enzyme. It cleaves peptides on the C-terminal side of lysine and arginine, except if they are followed by a proline.
• Chymotrypsin – catalyzes the hydrolysis of peptide bonds with tyrosine, phenylalanine and tryptophan.
• Pepsin – cuts after aromatic amino acids such as phenylalanine and tyrosine.
• Elastase – if the proteases above are comparable to “normal” restriction enzymes such as EcoRI, which cuts relatively rarely, elastase is your random cutter Sau3A. Elastase cleaves peptide bonds on the carboxyl side of hydrophobic amino acids such as alanine, glycine, and valine.

The enzymes above are just a first shot at studying your protein structure or posttranslational modifications. You can refine your protein analysis using more specific proteases.

References

1. Fontana A. et al. Probing protein structure by limited proteolysis. 2004. Acta Biochim Pol. 2004;51(2):299-321. [back]