As someone who has been working in assay design for many years while also running a contract research company that designs binding and activity measurements, I, along with others, have been getting more and more frustrated with commercially available protein reagents. At Affina Biotechnologies we buy purified proteins for our clients’ projects and a majority of these proteins have little material that is active in binding or enzymatic assays. These proteins are relatively inexpensive for small amounts needed to perform assays, so there is a very reasonable temptation to buy several from different providers and test them to find one that is of a reasonable quality. What we often see is that a lot of time and money is spent on testing these proteins only to discover that none are really active. It would have been cheaper to make the proteins from scratch and ensure that proper characterization and activity testing have been performed. 

Why does this happen? Genome sequencing coupled with modern molecular biology techniques enable rapid production of proteins, protein sub-fragments, and modified proteins. Companies that sell these count thousands of products. Yet, there are very few agreed upon standards by which these proteins are characterized that allow a customer to purchase a reagent and have it work in their system with any degree of confidence. A typical protein quality analysis is a test of its homogeneity by SDS-PAGE that is usually semi-quantitative at best. Occasionally, protein producers show a Western, usually using an antibody against a tag, which does not demonstrate the protein’s functionality in any way, and does not even prove the protein’s identity. There are many questions about a protein reagent that should be addressed by documents provided with commercially available proteins, but they are not.

What’s needed is a set of essential quality controls that a good reagent must have and the techniques used to characterize proteins when folded, as fully active proteins are needed. I won’t address characterization of post-translational modification of proteins, although that’s a fascinating and often essential aspect of protein characterization. The proteins pertinent to this discussion are needed for research involving measurement of enzymatic activity and binding properties, determination of physical protein characteristics including their three-dimensional structure, inhibitor screening and antibody production, among many others.  

In order to be confident in the quality of research reagents, we need assurances of the protein’s primary structure, presence and stoichiometry of co-factors, tertiary structure and functionality. Confirmation of the amino acid sequence and cofactor presence and stoichiometry can be readily and rather inexpensively determined by mass spectrometry, in some cases preceded by deglycosylation and aided by atomic absorption spectroscopy. The second level of physical characterization of a protein is determination of its tertiary structure. What is needed is the protein’s molecular weight determined by a variety of methods and the subunit composition determined by SDS-PAGE. Gel filtration chromatography provides a good compromise between precision and cost, especially when coupled with light scattering techniques. 

The final and essential hurdle in protein characterization is the activity test. Activity testing typically takes the form of an enzyme activity or a biological assay or an ELISA for antibodies. While these assays are very useful in demonstrating that some of the purified protein is active, they usually don’t show what fraction of the protein contributes the measured activity. Activity methods should be accompanied by measurements that effectively titrate binding sites and characterize this active portion in terms of the binding affinities of known substrates or ligands.

The life science community needs to resolve the issue of reagent quality; it is too expensive not to. Every day we waste time working with inactive, poorly folded proteins, contributing to the exorbitant cost of drugs and discoveries that were not made or, at the very least, were delayed. There are several possible solutions to the issue of reagent quality. If you need a protein and have the expertise and time, then you are the best person for the job. For those who have neither, there are high quality custom service providers that have the capacity to characterize proteins and measure their activity.  Once the large producers of reagents realize that the market requires a change in the quality of their products, they will be forced to change and sell reagents that are characterized with a range of techniques, perhaps creating a range of products with a different level of characterization. We just have to push them.

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Alexander Vinitsky, Ph.D.
Affina Biotechnologies, Inc.

Alexander was trained as an enzymologist at New York University. He studied the mechanism and functions of the proteasome during his tenure at Mount Sinai Medical School and made one of the first potent proteasome inhibitors. Alexander next moved to the pharmaceutical industry leading groups in biochemistry and molecular biology at Bristol Myers Squibb and Palatin Technologies. Since 2005 he founded two CROs. He is currently the president and chief scientific officer of Affina Biotechnologies, a CRO that specializes in assay development, screening and protein expression and purification.