Protein Expression
Services Provided by the Protein Expression Laboratory Core Facility
The goal of the UCLA-DOE Protein Expression Laboratory is to facilitate the cloning of genes, optimization of expression, and purification for the purpose of protein structure/function studies. It is the aim of the lab to collaborate with other researchers in order to facilitate protein expression for further structure/function studies.
Services include:
- Bacterial Expression: The Protein Expression Laboratory has the ability to clone genes directly from the organism or from a gene library, subclone into bacterial expression systems and to study expression levels. Several expression systems are currently being used, including pET, fusion systems, such as GST, His-tagged, MBP, protein A, and intein-CBP, as well as coexpression systems.
- Yeast Expression: Cloning or subcloning genes for expression in Pichia pastoris, Saccharomyces cerivisea, or Schizosaccharomyces pombe for protein expression.
- Protein expression studies: These studies are designed to optimize protein expression. Included, are the investigation of various strains (including protease deficient E. coli strains), media, growth and/or induction temperature, induction timing and levels, and/or coexpression (with chaperonins, thioredoxin, or tRNAArg).
- Fermentation: The Protein Expression laboratory is equipped with a New Brunswick BioFlow 3000 Fermentor which has the capacity to run up to 10L fermentations.
- Protein purification: These studies are designed to optimize protein purification. The optimal purification scheme is dependent upon the properties of the particular protein which is being purified. Procedures may be included, but are not limited to: French press, ammonium sulfate studies, addition of protease inhibitors, affinity chromatography (GST, amylose, metal chelate, heparin, dye, etc.), ion exchange chromatography, (anion or cation), hydrophobic interaction chromatography, hydroxyapatite, reverse phase and size exclusion.
- Fusion protein cleavage: These studies are designed to optimize cleavage of fusion proteins. Investigation of conditions may involve and exploration of temperature variation, additives (detergents, buffers, salts, cofactors, etc.), protease concentration, and timing of cleavage reactions.
- Crystallization screening: Optimal conditions for crystallization are very difficult to predict. An effective way to search through a large number of variables (pH, precipitants, salts, buffers, organics, etc.) which may effect crystal growth, is to conduct a sparse matrix search of specific crystallization conditions. We do this with commercial crystal screening kits (Hampton). This information can then be used to develop an expanded screen on subsequent studies.
- Protein production scale up: These studies are designed to optimize large scale protein production. In this case, protein expression and purification conditions have been established. This information will then be used to scale up protein production to the 10L or 170L scale.
