Instrumentation
The core facility houses a variety of equipment for cloning, expression and purification of bacterially expressed recombinant proteins; this includes vectors for ligation-independent cloning (LIC), numerous strains of E. coli for expression of challenging constructs and a refrigerated incubator for large-scale production of temperature-sensitive proteins. For large-scale purification, the lab has a GE AKTA Purifier FPLC system equipped with a variety of columns for size exclusion, affinity and ion exchange chromatography.
A SensíQ® Pioneer SPR system is available for characterizing protein-ligand interactions. The machine has three channels and includes an autosampler and two temperature-controlled slots for SBS-footprint deep-well blocks. This instrument offers a significant advantage over the more frequently used GE BiaCore models as it can derive kinetic parameters from single "OneStep" injections.
The core is equipped with a Rigaku MicroMax 007-HF X-ray generator and a Rigaku R-AXIS IV++ image plate detector. Cryo-equipment includes an Oxford Cobra cryostream, an inverted phi axis for easy retrieval of cryo-cooled crystals from the X-ray goniometer head and a SAM cassette for long-term storage of crystals under cryo-conditions. For experiments which may require a more intense X-ray beam, such as for high throughput data collection, or a tunable X-ray source, the facility also has rapid access to the SSRL synchrotron at Stanford University.
Two ARI robotic systems have recently been added to the core facility; a Gryphon for nano-liter liquid handing of SBS-footprint plates, and a Scorpion for custom liquid handling. The former is primarily used for setting up protein/DNA crystallization trials - using as little as 20 μL to screen 96 reagent conditions. The Scorpion is a more versatile robot which is principally used in optimization of crystal "hits." Two incubators set at 4°C and 18°C are dedicated for housing crystallization plates.
Microscale Thermophoresis (MST) is the next generation of technology to screen drug against protein target.
The instrument will complement surface plasmon resonance technique available in the Molecular Therapeutic Core. In particular, MST has capabilities to measure ligands/drugs for difficult drug targets such as transcription factors and membrane proteins. The key feature of this technology is the ability to measure affinity either using a recombinant proteins or proteins expressed in a cell lysate with greater precision.
In addition to the equipment mentioned above, the molecular therapeutics core facility includes GE MicroCal calorimeters for thermodynamic analysis of protein-ligand interactions through ITC. A Waters Breeze 2 HPLC equipped with a number of columns for small molecule and protein chromatography is also available for use.
Contact the Molecular Therapeutics Research Core
8700 Beverly Blvd.
Davis, 5007
Los Angeles, CA 90048