SCIENTIFIC CORES
Bacterial Protein Expression Core
Biochemistry Core Cell Biology and Imaging Core Computation Core EM Crystallography Core EM Tomography Core Eukaryotic Protein Expression Core Fluorescence Spectroscopy Core Protein Interactions Core Protein NMR Spectroscopy Core RNA Structure and Dynamics Core Tissue EM Core Virology Core Virus Imaging Core X-ray Crystallography Core |
Eukaryotic Protein Expression Core
Director: Wes Sundquist, PhD Manager: Steve Alam, PhD Proteins and protein complexes that cannot be expressed readily in bacteria are produced in one of two eukaryotic protein expression systems: baculovirus/insect cells, or cultured mammalian cells. Both systems are used to produce milligram quantities of proteins and complexes of interest. Laboratory Facilities Eukaryotic protein expression is performed in our BSL-2+ tissue culture laboratory (350 sq. ft.). The laboratory is fully equipped for tissue culture, with tissue culture hoods (3), refrigerators (2), chilled centrifuges (2), incubators (5), liquid nitrogen storage freezers (2), and microscopes (1). Insect cultures are grown in dedicated stationary incubators (2) and rotary shakers (4) within an adjacent dedicated temperature controlled room. Baculovirus/Insect Cell Protein Expression Both baculovirus and insect cell expression systems are currently being used for high-level expression of recombinant proteins. Proteins such as primate TRIM5α that are the focus of intensive study are typically expressed using a baculovirus system in order to maximize yields. Briefly, the BaculoDirect system (Invitrogen) is used to produce recombinant baculoviral stocks, which are propagated and amplified in SF9 and SF21 insect cell lines. Optimal protein expression is then typically achieved in specially engineered SF9 and High Five (Invitrogen) suspension cell lines. Multi-protein complexes are currently produced by co-infection of target cells with relevant baculoviral constructs at optimized MOIs. In cases where multiple constructs must be screened rapidly, we employ two different methods. Often, we use the Invitrogen Drosophila Expression System (DES), which allows for direct transient transfection of protein expression vectors into Drosophila S2 cells. This method is used to produce small to medium scale quantities of multiple different protein constructs (e.g., mutants and fragments for crystallization) because it is considerably faster than baculovirus production. We are now moving to the pFastBac baculoviral system (Invitrogen), which can produce high viral titers in half the time of the BaculoDirect system while still using a viral system for high levels of protein production. Proteins and complexes are typically expressed with epitope tags to facilitate protein purification. OSF (One-STrEP-FLAG) serves as our primary affinity purification tag, but we also have access to expression vectors with C-terminal FOS, (His)6, GST, MBP, 3X FLAG, Myc, VSV-G, HA, CBP, and V5 tags. Protein Expression in Mammalian Cell Culture Large-scale recombinant protein expression of proteins and complexes in mammalian cell culture is performed by transient transfection into the highly transfectable cell line HEK293T (Human embryonic kidney cells). To minimize cost, polyethylenimine transfection protocols are being used for both monolayer adherent and suspension cultures (in spinner flasks and shaker flasks), with ~90% transfection efficiencies. Recombinant proteins are expressed using a series of different vectors (pcDNA-DEST40, pCAG-OSF, pEF-OSF and pcDNA-FOS) that feature efficient promoters and various affinity purification fusion tags. Protein complexes are produced by optimized co-transfection of multiple plasmids, an approach that we have used successfully for complexes such as human ESCRT-I. Alternatively, where proteins (or complexes) may express better in mammalian cells than in insect cells, we are now beginning to test baculoviral vectors that are also capable of expressing proteins in human cells (e.g., the BacMam vectors, see Dukkipati et al. “BacMam system for high-level expression of recombinant soluble and membrane glycoproteins for structural studies” (2008) Protein Expr. Purif. 62:160-170). Protein Purification Proteins are typically purified using Streptactin affinity chromatography followed by conventional chromatography on one of 4 FPLC systems (two Äkta Primes, one AKTApurifier and one ÄKTAxpress (GE Healthcare)). During the current funding cycle, we plan to obtain another ÄKTAxpress Chromatography System, which will be shared equally with the Bacterial Protein Expression Core.  Steve Alam (Manager)  |