High-Throughput Protein Crystallization and Crystallography Services

SARomics Biostructures is a contact research organization (CRO) that offers protein crystallization, protein crystallography, and protein-ligand complex analysis services to pharmaceutical and biotech companies as well as to academic groups. Crystallization and crystallography are the oldest methods for three-dimensional structure determination at atomic level. After the discovery of X-rays by Conrad Röntgen (Nobel Prize in Physics, 1901), the basic principles of X-ray crystallography were discovered by Max von Laue (Nobel Prize in Physics, 1914) and the Braggs father and son (Nobel Prize in Physics, 1915). Currently, X-ray crystallography and protein NMR are the only two methods, which provide atomic resolution tertiary protein structures for structural biology. The majority of entries in the Protein Data Bank (PDB) have been determined by X-ray crystallography and NMR spectroscopy.

1. Protein expression, purification and characterization
The company’s
custom protein and protein-ligand crystallization and X-ray crystallographic structure determination services (fee-for-service crystallography) include all steps required for successful protein crystal structure analysis. Within our gene-to-structure services, after cloning, expression and purification, and prior to crystallization, an accurate characterization of the state of the protein in solution is performed. Probably the most important requirement is a monodisperse solution of the protein, which should not contain aggregated or denatured material, and is preferably stable, for example, within a certain range of pH and ionic strength (these values will be varied during crystallization screening). By other words, a proper characterization of the protein solution is critical for the success of the whole project.
Our
services platform is built up around the most advanced biophysical methods for protein characterization, and includes dynamic light scattering (DLS), CD spectroscopy and differential scanning fluorimetry (DSF, similar to thermal-shift assay. The method is frequently used at the Structural Genomics Consortium for protein characterization prior to crystallization). Protein NMR spectroscopy is also used if required, for the initial verification of target constructs and to answer the question: Does the construct result in a folded, well-behaved protein? The HSQC fingerprint spectrum of the protein readily shows whether the protein is well-folded, unfolded or ‘molten-globule’ like, and whether parts of the protein are flexible. This information is often very useful as a preparatory step before initiating crystallization trials.


2. Protein crystallization
Protein crystal structure determination requires crystals. After protein characterization, conditions for obtaining well-diffracting single crystals need to be identified (if the protein has not been crystallized before). The number of experimental parameters affecting protein crystallization can be very large. Among these is the type of the buffer and the pH of the solution, the ionic strength, the presence of various salts, the temperature of the experiment and the presence of various ligands (co-factors, substrate analogues, inhibitors, etc.). In addition, we need to identify the most suitable type of precipitant, which is one of the most central components in the setup.

The most popular precipitants used in protein crystallography include polyethylene glycols of various lengths, ammonium sulfate, and some alcohols. To find the right combination of parameters, one needs to screen a large number of different conditions. Several hundreds, and often thousands of different conditions are screened before the protein crystallization conditions can be identified. Commercial screens, like those from
Hampton Research or Molecular Dimensions, are initially used. However, we have the facilities to design our own screens, if required (usually done during optimization of the conditions found using the initial screens).

In
protein 3D structure determination for the most efficient identification of the crystallization conditions, we use high throughput and high precision screening and imaging robotics. For example, 96 different crystallization conditions can be screened with as little as 15 microL of protein sample using a Mosquito liquid handling robot. The use of robotics helps in saving both time and precious material. When an initial condition is found, it normally needs to be optimized further to yield crystals more suitable for X-ray crystallography.

Our
protein crystallography services platform includes FastLane Premium and FastLane™ Standard libraries. In the case of the FastLane Premium library, we already have crystallization-grade protein stored in our freezer ready for crystallization set up. For the FastLane™ Standard library, we have stored constructs with established and verified protocols for expression, purification and crystallization.
In addition, using our
Gene-to-Structure plattform we will carry out cloning, expression, purification, crystallization and structure determination by X-ray crystallography or NMR spectroscopy of any protein requested by a customer.

The combination of FastLane structures with fast access to protein crystallography synchrotron beamlines (we collect X-ray data every second week) and our adapted high-throughput crystal structure solution protocols help us minimize the time between the very first meeting with a new customer to the delivery of the final protein structure or a ligand complex.