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 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.
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 screening uses bacterial (E. coli) and yeast (P. pastoris) 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 accessory plasmids (pLysS, pRIL, pRARE, etc), and lysis buffers.
LARGE SCALE FERMENTATION AND CELL LYSIS
After suitable protein expression/solubility conditions have been determined this information is used to scale up cell growth to increase recombinant protein yields. The PETC is equipped with a New Brunswick BioFlow 3000 fermentor and three BioEngineering NLF22 fermentors which have the capacity to run fermentations of ~12.5L. The PETC is equipped with a French press and Avestin Emulsiflex C-3. The Emulsiflex is particularly well suited for efficient high-throughput cell lysis with a throughput of 50 ml of lysate per minute.
Using predetermined information on protein expression and solubility from small-scale experiments recombinant proteins are purified in large-scale using BioRad BioLogic chromatography systems. Chromatography methods include: affinity chromatography (GST, amylose, metal chelate, heparin, dye, etc.), ion exchange chromatography (anion or cation), and size exclusion. If required fusion proteins and/or affinity purification tags can be proteolytically cleaved and removed at this stage.