Section 3.1.6.2. Protein storage

Even when the efforts of those engaged in crystallization and those engaged in producing the desired recombinant protein are well coordinated, it is not usually appropriate or desirable to use all the available protein for crystallization at the same time. This means that some of the material must be stored for later use. Even under the best of circumstances, protein solutions are subject to a number of unwanted events that can include, but are not limited to, oxidation, racemization, deamination, denaturation, proteolysis and aggregation. As a general rule, it is better to store proteins as highly purified concentrated solutions. This reduces problems of proteolysis (since the proteases have been removed), and, in general, proteins are better behaved if they are relatively concentrated (greater than 1 mg ml⁻¹). This is not an absolute rule, however; if there are problems with aggregation, these can sometimes be minimized by storage of proteins in dilute solutions, followed by concentration of the samples immediately prior to crystallization. If the protein contains oxidizable sulfurs (free cysteines are a particular problem), reducing agents can be added (and should be refreshed as necessary), and the solutions held in a non-reducing (N₂) atmosphere. In some cases, it is easier to mutate surface cysteines to produce a more stable protein (see Chapter 4.3 ).

In general, proteins behave best under conditions of pH and ionic strength similar to those they would experience in the normal host. Usually this means a pH near, or slightly above, neutral and intermediate ionic strength. These conditions are often not the ideal conditions for crystallization, and dialysis or other forms of buffer exchange may be required before beginning crystallization trials. In general, protein solutions are stored either at 4 °C in a cold room or refrigerator, or at 0 °C on ice. It is essential that the protein be stored in a manner that will not allow microbial growth, usually achieved by sterilization of the protein solution by filtration through 0.2 micron filters and/or addition of antimicrobial agents, such as . For long-term storage (periods longer than a few weeks), protein solutions are often precipitated in ammonium sulfate or frozen at either −20 or −70 °C. Repeated freezing and thawing is not recommended; if a protein sample is to be frozen, it should be divided into aliquots small enough so that each will be thawed only once. Whenever a protein sample is frozen and thawed, some loss of quality and/or activity can be expected. Freezing samples of intermediate concentration (1–3 mg ml⁻¹) usually works better than freezing either extremely dilute or concentrated samples. Cryoprotective agents can be added to protein samples destined to be frozen; however, it should be remembered that the same reagents that are helpful when freezing a protein sample may be distinctly unhelpful when that sample is thawed and used for crystallography. Most biochemists willingly add glycerol to their protein samples before freezing; crystallographers are not usually happy to find that their protein sample is dissolved in 50% glycerol. Both pH and ionic strength can affect a protein's tolerance to freezing and thawing. In many cases, buffer exchange and concentration procedures need to be performed to convert stored protein solutions to ones suitable for crystallization.

As is so often true in science, decisions about whether to freeze a particular protein sample and, if it is to be frozen, exactly how the freezing should be done, depend on experience. If the protein in question is an enzyme, it is often useful to set up a series of trials in which small aliquots of the protein are stored under a variety of conditions. If the aliquots are tested on a fairly regular basis, how stable the protein is in solution can usually be determined, as well as how well it will tolerate a cycle of freezing and thawing, with or without an added cryoprotectant. If enzyme assays are not available, other methods of characterization, such as gel electrophoresis, mass spectrometry and light scattering, can be used to check for degradation, oxidation of cysteines and aggregation. Armed with this information, and with a plan for how the protein will be used for crystallization, it is usually a fairly simple matter to decide whether or not to freeze a particular sample, and, if the sample is to be frozen, how best to do it. It is a good idea to make such tests early in a major crystallization effort. This will avoid the awkward dilemma that occurs when a large amount of a highly purified protein is available, and the knowledge of how best to store it is not.