Overexpression of recombinant proteins in any of three systems
We provide consultation for all projects and can assist with the design, construction, and expression testing of vectors featuring various affinity tags (i.e., His, GST, FLAG) and/or fusion partners (i.e., TRX, MBP, EGFP) in order to maximize the expression level of the target protein in E. coli.
Depending on the nature of the heterologous target protein, we provide and utilize a number of different E. coli host strains to improve expression levels and to maximize the odds of proper folding. For example, Rosetta™ cells are used for protein constructs containing a high number of rare E. coli codons, and Origami™ cells are used to encourage proper disulfide bond formation.
We maintain and utilize common proteases, such as TEV, PreScission and Sumo. If there are concerns about affinity tags or fusion partners interfering with the structure and function of the target protein, we can provide reliable methods for removing affinity tags.
In some cases, especially during overexpression in E. coli, expressed protein accumulates as insoluble inclusion bodies and several strategies can be used to improve the solubility of the expressed protein. Our core is equipped with Thermo MaxQ high performance shakers for optimization of expression temperature to maximize soluble expression.
For proteins that express insolubly, we can provide purification under denaturing condition (e.g. in high concentrations of urea). We are experienced in using DSF guiding refolding to determine suitable conditions to refolding by which to produce properly refolded protein from insoluble inclusion body preparations. We also routinely produce small peptides via insoluble expression in E. coli.
Insect Cells/Baculovirus Expression System
We construct recombinant baculoviruses with expression DNA vectors/bacmids provided by the investigator. Insect cells are co-transfected with wild type Autographa californica nuclear polyhedrosis virus DNA (AcNPV) and baculovirus transfer plasmids and isolates recombinant baculoviruses that arise by homologous recombination. Recombinant baculoviruses can also be generated by transfection of bacmid DNA using the Bac-to-Bac system. Recombinant baculovirus stocks from the transfection or co-transfection are plaque purified by an agarose overlay assay. Individual recombinant viral isolates are plucked from agarose and used to infect Sf9 insect cells. The infected Sf9 cells (for intracellular proteins) or culture supernatant (for secreted proteins) are provided to the investigator for expression screening and identification of the isolate with the highest level of expression. Screening is typically done by immunoblot assay.
We titer and amplify chosen positive recombinant baculovirus isolates. After selection of a specific recombinant viral isolate, a passage #1 seed stock is expanded to a 500ml volume and the passage #2 viral stock is titered by an agarose overlay plaque assay to determine the virus concentration in pfu/ml. Small aliquots of the passage #2 virus are placed at -70°C for long-term storage. The bulk of the amplified 500ml virus stock is stored at 4° C for use in protein production. In addition to amplifying seed viral stocks from newly generated recombinant baculoviruses, the resource also amplifies, titers and stores viral stocks from existing recombinant baculoviruses provided by investigators.
We express proteins of interest(POIs) in insect cells using recombinant baculovirus. Protein production is performed on a small scale in conventional spinner culture vessels in the range of 50-500volumes or on a large scale in oxygenated 5-liter bioreactors. The resource maintains two insect cell lines for protein production,Sf9 and High Five cells. The Sf9 cells are typically used to express intracellular proteins, while High Five is often preferable for secreted proteins. The expressed protein is provided to investigators either as a washed cell pellet (intracellular proteins) or as a culture supernatant (secreted protein) after pelleting of cells by centrifugation. The Core can also purify the protein of interest, if desired. Insect cells for protein production are typically grown in Grace's Insect Medium supplemented with 10% FBS(Sf9) or Express Five chemically defined serum free media (HighFive). If requested by an investigator, Sf9 insect cells will be adapted to a serum-free defined medium.
HEK293 Cells(Transient transfection)
We use Thermo/Invitrogen Expi293 expression system. The Expi293 Expression System is a complete solution for rapid, high-yield protein production from mammalian 293 cells. It is based on integrated components including high-density Expi293F cells in Expi293 Expression Medium and the cationic lipid-based ExpiFectamine 293 transfection reagent in combination with specialized transfection enhancers. The Expi293 Expression System features:
- Up to 1 g/L of protein; 2-to 10-fold higher protein yields than previous generation transient expression systems such as the FreeStyle 293 Expression System
- Rapid production of recombinant proteins in 5–7 days
- High density cultures of Expi293F cells that result in more expressing cells per milliliter of culture
- Native folding and mammalian post-translational modifications of expressed proteins
- Easy, robust culture and transfection protocols
- Easy to upscale to liters