Structure Based Design, Inc.

SBD's Technologies

Molecule Cloning

Production of large quantities of protein samples with high quality is essential for carrying out a high quality crystallographic study. To find the best protein construct and the highest resolution crystal for a client’s project, we generally design and express multiple constructs of the target protein for co-crystallization study.   The gene of the target protein will be prepared using a chemical synthesis technique. The method has become a powerful molecular tool for optimizing protein expression. An optimal DNA sequence can reduce secondary structures, contrary repeats and repetitive sequences. The expression vectors we used are optimized for a high level expression. We have had excellent experiences in expressing difficult proteins, such as nuclear receptors, membrane associated proteins and Ion Channel proteins. 

Protein Expression and Purification

Protein samples will be expressed using an E. Coli expression system or baculovirus expression system.  We have state-of-the-art fermentation systems and bioreactors for production of large quantity of recombinant protein using E. coli, and baculovirus-insect cell in-house.

Our senior experts have had over a decades’ worth of experience in cell culturing. The in situ 3 liter Fermenter is especially suited for large scale protein production. Unlike cell culture via flask, Fermenter allows cell growth at a high density. The final cell density can be hundreds of times more than the production using a flask. 

We have two Akta Explorers and one Akta Purifier FPLC systems with numerous large working size columns (affinity, ion exchange, hydrophobic, sizing, desalting). By combining Fermenter and liter sized chromatography systems, we can produce quantities of proteins ranging from 10 mgs to a few grams. This is a great advantage for preparing large quantities of protein for different assays or crystallography studies, particularly for a large project.   All protein samples prepared in our laboratories for crystallization study will be tested for quality assurance. The purity and activity of the sample will be reported to our clients. The sample also can be tested in a clients’ laboratory using different assays.

 

Protein Crystallization and SBD’s Proprietary Crystallization Techniques

We have developed a screen kit that has four hundred non-overlapping crystallization conditions to carry out high throughput crystallization screening. To obtain good crystallization conditions, we use different approaches including vapor diffusion crystallization, batch crystallization, growing crystals in Agarose gel, seeding, cross-seeding and crystal re-growth techniques to find the crystallization conditions and optimize crystal quality.

Determine the highest resolution crystal structure

Our mission is to grow the best quality, highest resolution crystals for clients. Crystallization and co-crystallization are key steps in our services. We have developed multiple proprietary technologies and have formed an efficient platform to provide highly qualified services. Our innovative techniques are developed for different issues of protein crystallization including stimulating nucleation, control crystal number, crystal quality selection, fast crystal growth, improved crystal stability in a soaking solution and methods of handling hydrophobic compounds. The crystallization and co-crystallization methods are our most valuable core techniques. They form our distinct strengths to perform our services. Our aim is to be the best vendor in this field by demonstrating to clients our superior techniques and services.

We want the crystal structures from our hands to reach the highest caliber. We do not care how much protein sample will be used, or how many trials needed to reach this goal. We have achieved our promise in all projects we have ever worked on, such as a 1.5 Å HCV NS5B co-structure, 1.6 Å FXR co-structure, 2.0 Å A-site RNA co-structure, 1.6 Å Androgen Receptor co-structure, 2.3 Å co-structure of a human Aurora-A construct et al.

Determine native conformation of a client’s protein

It has been well known that protein conformation may be sensitive to the crystallization solution. To guide drug design with the native conformation of a protein, we have developed our proprietary crystallization screen kit, “Native Conformation Screen™”, for drug target protein crystallization. We will not use high concentrations of salt and organic reagents in our crystallization screen. The reagents we use in the crystallization trials have soft features for the protein which ensure that it will maintain its native conformation. We always do our best to find co-crystallization conditions that are close to the physiological conditions of a protein.

Determine protein conformation of different active stages

To show detailed pictures of how the target protein and inhibitor interact, SBD scientists not only work on single protein crystal structures but also try to get crystal structures of different conformational states of a protein, such as the inactive form and full active form of a kinase, and a nuclear receptor with or without co-activator. 

Determine co-crystal structures rapidly

Once the first batch of co-crystal structures is determined, SBD’s scientists will analyze the co-crystallization data and characters of a client’s compounds to design a more efficient approach to generating co-crystals for additional compounds. After solving several co-crystal structures, we will be able to establish a specific protocol for a client’s compounds. The protocol will streamline the protein preparation, co-crystal generation, co-crystal structure determination and refinement processes for a client’s project.  We expect to generate co-crystal structure in less than one week using the soaking technique or less than two weeks using the co-crystallization technique with this protocol. This fast co-crystal structure determination service is known as the “Co-crystal Structure Express” approach.

X-ray Data Collection

We will test crystal diffraction with local X-ray equipment and collect data sets using Synchrotron Light Source.

To support the research of crystal structures at SBD, We maintain agreements that enable us to  rapidly access the Advanced Photon Source (APS) at the Argonne National Laboratory (http://www.aps.anl.gov), the Advanced Light Source (ALS), a division of Berkeley Lab (http://www-als.lbl.gov) and Stanford Synchrotron Radiation Laboratory (SSRL), and Stanford University (http://www-ssrl.slac.stanford.edu). The laboratories provide very bright and tunable X-ray sources which allow us to solve new crystal structures with the MAD method and collect the data set with the highest resolution. We are also in contact with synchrotron authorities for "instant access" to the Beamline to match the urgent need for co-crystal structures.

Structure and Co-structure Determination

Crystal structures will be solved with the Molecule Replacement method or Multi-wavelength Anomalous Diffraction (MAD) technique.  The co-crystal structures will be determined with the high resolution data set collected from the synchrotron light source. The quality of the final X-ray structure should match the publication requests for a crystal structure. Our crystallographers have had experience to solve various drug target proteins including kinases, nuclear receptors, polymerases, dehydrogenases, Proteases, RNA et al.

Location and refinement of client’s compound is an essential component of a co-crystal structure. SBD will provide the highest quality co-crystal structure that the drug target can reach. The electron density map of the ligand in the complex structure should be well refined in order to determine the position of all atoms.