Experimental enzymology - From production to assays

Since the early 1990s, remarkable progress has been made in a number of scientific fields:

• Molecular biology, with its knowledge of genomes, cloning, experimental techniques and the development and use of vectors;

• microbiology, with the determination of various host strains and culture media;

• biochemistry, with the development of chromatography columns;

• biophysics, with the possibility of coupling surface plasmon resonance or two-dimensional gel electrophoresis with mass spectrometry to optimize the production and purification of proteins and enzymes;

• accelerated determination of the spatial structure of proteins and enzymes using artificial intelligence applications.

As a result, access to a better understanding of enzymes and optimization of their production and purification have become more accessible.

At the same time, improvements have been made in assays for measuring enzyme activity, and in assays for qualifying and quantifying the binding between an enzyme and an effector.

These techniques are increasingly resolute, reliable and environmentally-friendly. They have accelerated research in various therapeutic fields. A case in point is the remarkably rapid development of kinase inhibitors in oncology, thanks to the rapid genesis of chemical or biological molecules, coupled with equally rapid, highly resolving target testing. Whereas thirty years ago, there were virtually no therapeutic research projects targeting these enzymes, today there are so many that access to all marketed drugs targeting kinases, among others, is becoming more difficult. Against this background, this review offers a non-exhaustive overview of techniques and protocols for purifying and measuring enzyme activity.

At the end of the article, readers will find a glossary and a table of acronyms and notations.