Recombinant Protein Production, Characterization, and Crystallography core

Expression of Monoclonal Antibodies (Mabs) from Existing Hybridomas (In Vitro)

Expression of Mabs from hybridoma cell lines is provided in spinner vessel cell cultures (0.5 to 3 liters) in medium containing low bovine IgG serum or in chemically defined serum-free medium.   Culture supernatants from spinner vessels produce Mabs in the range of 5-50 mg/liter. Larger amounts of Mab can be produced in CL-1000 Celline flasks with the ability to generate antibody in the range of 50 -300 mg/liter. 

Purification of Monoclonal Antibodies  

MAbs expressed from hybridomas in culture are typically purified by Protein G/A affinity chromatography but can also be purified by custom ion exchange and size exclusion chromatography procedures.  Analysis and Q/C of purified Mabs is provided by SDS-PAGE for purity, protein concentration by NanoDrop and analytic size exclusion chromatography to determine homogeneity of native molecular size. Endotoxin-free purified Mab can be provided upon request.

We are equipped with GE Healthcare’s ÄKTA Pure and ÄKTA Start chromatography systems, which allow for unattended operation and the efficient automation and optimization of purification tasks.

Choices of Chromatography Techniques

Commonly used chromatography techniques include: size exclusion chromatography (SEC, i.e., gel filtration), ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and affinity chromatography.

Affinity chromatograph is usually combined with specific affinity tags (i.e., 6xHis, GST, FLAG, etc.) that are used for the first step of purification to achieve enrichment of the target fusion protein.

Ion-exchange chromatography (IEX) step is usually chosen as a second binding/elution process of purification. Depending on the pI-values and differences between target and contaminant proteins, anion-exchange or cation-exchange matrices can be used to further purify the desired protein.

Gel filtration chromatography (SEC) is usually performed as a final polishing step and can be used to increase the purity and homogeneity of the protein sample. In brief, the proteins within a sample are separated on a porous matrix according to their size. Thus, not only can contaminant proteins with different molecular weights than that of the target protein be separated, but different oligomeric forms of the target protein can also be resolved. This polishing step can be very critical, especially for structural protein studies.

Gel filtration can also provide valuable information for the analysis of protein-protein interactions and can be informative about the association between one or more proteins or nucleic acids, under diverse conditions.

Prepacked Columns

We carry GE Healthcare’s prepacked affinity columns (i.e., HisTrap, GSTrap), desalting columns, and all sizes of SEC columns (from analytic scale Superdex increase to production grade Superdex). We also have hydrophobic interaction columns for procedures such as the removal of endotoxin and nuclease contaminants.

Overexpression of recombinant proteins in any of three systems

E. coli

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

We provide various protein characterization technologies and instruments.

Instruments are available in the Core to characterize proteins and other macromolecules. Interactions between compounds can be detected and analyzed using ITC. BLI OCTET can be used for high throughput binding affinities and kinetics. MALS will be available to assess a protein’s hydrodynamic size, size distribution, and aggregate population. Protein stabilities can be measured by DSF.

Currently available instruments, please schedule through iLab.

• Octet
• AUC (Analytical Ultracentrifugation)
• Auto-ITC (Isothermal Titration Calorimetry)
• DLS (Dynamic Light Scattering)
• DSF (Differential Scanning Fluorimetry) Roche Thermal Cycler
• SEC-MALS (Size Exclusion Chromatography- Multi-Angle Light Scattering)

Macromolecular X-ray Crystallography services provide access to this advanced technology and assist researchers in determining the structure of their macromolecule of interest. In addition, we offer structural model building using AlphaFold and virtual drug screening services based on structural models and compound libraries.