Sample Production

Prior to starting the crystallization process, researchers must have pure macromolecular samples. The best macromolecular samples for crystallization experiments are homogenous in content, chemistry and conformation. For proteins, recombinant methods are most often employed, but there is a bewildering number of alternative protocols to consider before starting.

Realizing that some investigators' labs are poorly equipped — and that investigators may have limited expertise — the X-ray Crystallography Core provides infrastructure, methodology, advice and some supplies to produce the samples needed. The core can also be a collaborative resource for projects that require pure, intact protein for experimental purposes other than crystallography.

Over the past two decades, projects involving James R. Thompson, Ph.D., core director, have expressed and purified numerous functional proteins from bacteria, yeasts and insect cells. Because sample production is often difficult, many different technologies and strategies have been employed to optimize successful yield along with ease, speed, cost and versatility.

Higher throughput protein expression methods developed by structural genomics centers are now being implemented, as crystallization results using only one sample are more uncertain. Additionally, completely novel protocols are being developed for easier and less expensive expression or co-expression of difficult-to-make eukaryotic proteins.

For example, the core is developing innovative high-yield methods to express or co-express integral membrane, extracellular and intracellular proteins within silkworm larvae or pupae at one-third the effort and one-sixth the cost of conventional insect or mammalian cell culture protocols.

The core can also use DNA for crystallization trials, though the abundant and highly purified oligo samples required must be manufactured elsewhere.

With samples in hand, researchers can move on to crystallization.