• Computer Core
• Crystallization Core
• Protein Expression
• X-ray Crystallography
• NMR Core
• Instrumentation

Protein Expression

The Protein Expression Technology Center (PETC) was founded in 1994 to facilitate the expression and purification of proteins for structure/function studies. The PETC provides support in all aspects of protein expression from cloning through expression optimization and protein purification.  The PETC is a UCLA-DOE Institute for Genomics and Proteomics facility but is open to all UCLA researchers.

The evaluation of many expression constructs can be a crucial step in obtaining a protein sample suitable for further characterization. Recent advances in cloning and protein expression based on technologies developed by structural genomics consortia have resulted in the ability to evaluate tens or hundreds of expression constructs rapidly and economically. We have adapted these techniques to allow for a medium throughput approach suitable for the hypothesis-driven projects of academic research groups. Our resources allow rapid analysis of conditions that yield well-behaved soluble protein for downstream biochemical and biophysical studies. 

Services Provided by the Protein Expression Technology Center Core Facility

Services include:

• Cloning and construct design: The PETC clones genes directly from an organism or library using conventional restriction enzyme mediated, ligation independent cloning (LIC), and enzyme-free cloning (EFC) techniques. LIC and EFC techniques allow expression constructs to be rapidly and economically generated. Bioinformatics data is used to guide the design of expression constructs.

• Bacterial and Yeast Expression: Bacterial expression systems used by the PETC include a variety of inducible protein expression vectors (pET and pBAD), fusion systems (GST/His6/MBP/NusA/Thioredoxin) for increased solubility and affinity chromatography, and vectors to co-express chaperones or rare tRNAs to promote folding and increase recombinant protein yields.  Yeast expression systems use constitutive or inducible promoters and allow for secreted or cytoplasmic expression of target proteins.

• Protein expression screening: Protein expression studies use bacterial (E. coli) and yeast (P. pastoris, S. cerevisiae, or S. pombe) expression hosts. A ShelLab SI6R-HS shaking incubator allows growth of up to 1152 cultures in 96-well format to high culture densities. The high cell densities allow microgram amounts of recombinant protein to be produced in the small culture volumes used in 96-well plates. Cell lysis is performed in 96-well format and SDS-PAGE analysis is used to evaluate target protein expression. Batch purification of protein from the soluble fraction is used to assess the ability of the target protein to bind affinity purification resins.  Cell growth in 96-well format allows parallel investigation of parameters critical for obtaining high yields of recombinant target proteins.  Parameters explored include: expression strains, media, growth and/or induction temperature, coexpression with chaperonins or tRNAs for rarely used codons, and lysis buffers.

• 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 exploration of temperature variation, additives (detergents, buffers, salts, cofactors, etc.), protease concentration, and timing of cleavage reactions.

• Large scale fermentation: After protein expression and purification conditions have been established this information is be used to scale up cell growth to increase recombinant protein yields. The PETC is equipped with a New Brunswick BioFlow 3000 Fermentor which has the capacity to run up to 10L fermentations.

• Crystallization screening: Optimal conditions for protein crystallization are 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. Commercial crystal screening kits (Hampton Research or QIAGEN) are used to identify initial crystallization leads.  Initial “hits” are used to design optimization strategies for expanded screens to obtain diffraction-quality crystals for structural studies.