Recombinant Protein and Monoclonal Antibody Production
(Dean Edwards, Ph.D., Director)
Recombinant Protein Expression (Baculovirus system)
- Construction of Recombinant Virus: Insect cell cultures are 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 viruses in the culture supernatant of the transfected cells are plaque purified by an agarose overlay assay. Individual recombinant viral plaques are then plucked from the agarose, extracted, 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 to screen for virus that produces intact protein. Screening is typically done by immunoblot assay or by gene expression.
The investigator is required to clone the cDNA of interest into an appropriate baculovirus transfer plasmid. The Core does not construct recombinant transfer plasmids, but does provide wild type viral DNA and transfection reagents. Numerous transfer plasmids with convenient restriction sites for cloning are available commercially. We will provide consultation on choice of transfer plasmids.
- Amplification and titering of recombinant baculovirus stocks: After selection of a specific recombinant virus, a passage #1 seed stock is prepared to a 500ml volume and a passage #2 viral stock is tittered as pfu/ml by an agarose overlay plaque assay. 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. In addition to amplifying seed viral stocks from newly generated recombinant baculoviruses, the Facility also amplifies, titers, and stores viral stocks from existing recombinant baculoviruses provided by investigators.
- Protein Production in Insect Cells: Protein production is performed on a small scale in conventional spinner culture vessels in the range of 150-1,000 ml volumes or on a large scale in oxygenated 5 liter bioreactors. The Facility maintains three insect cell lines for protein production including Sf9, Sf21 and High 5. The Sf9 cells are typically used to express intracellular proteins, while High 5 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. Insect cells for protein production are typically grown in Grace's Insect Medium supplemented with 10% fetal bovine serum. If requested by an Investigator, the insect cells will be adapted to a serum-free defined medium.
Hybridoma/Monoclonal Antibody Production
- Construction of New Hybridomas: The technical staff performs mouse immunizations and all cell culture procedures required to construct antibody secreting hybridoma cell lines. The investigator must assay mouse serum for specific antibody response. Specific procedures include removal of mouse spleen, PEG-mediated cell fusion with mouse myelomas, propagation of hybridomas in HAT selection medium, initial ELISA screening of hybridoma fusion wells, subcloning of positives by limiting dilution in 96 well microtiter plates, and expansion and freezing of clones in liquid nitrogen. Additionally, the Facility performs isotyping assays of cloned cell lines. Investigators are asked to perform secondary screening assays to confirm specificity of the hybridomas selected by ELISA. Secondary screening typically involves immunoblotting, immunoprecipitation, immunocytochemistry or functional effects on the antigen. Several mouse myeloma cell lines are maintained as fusion partners including SP 2/0, P3-X63-AG8.653, NS-1, and FOX-NY.
- In Vitro Production of MAbs from Existing Hybridomas: The facility mass produces monoclonal antibodies(MAbs) in vitro by three methods:
* Conventional spinner vessels in volumes that range from 0.5 to 5 liters. Investigators are provided with culture supernatant as a source of MAbs with yields ranging from 5 – 50 mg/ml.
* Stationary membrane bioreactor system (CELLine CL-1000, BD Biosciences). The level of production by this system is intermediate between the hollow-fiber perfusion system and conventional spinner vessels, yielding antibody concentrations in the range of 15 - 250 mg/ml.
* Hollow-fiber bioreactors. The cells grow to a high density on the large interior surface area of the capillary fibers and are capable of secreting high concentrations of MAbs in the range of 0.5-1.5mg/ml.
- Down–stream processing and purification of MAbs: The facility will purify MAbs produced in vitro by any of the above procedures by standard methods.
* Ammonium sulfate precipitation followed by affinity chromatography on Protein A/G agarose resins or DEAE ion-exchange.
* Analysis of purified MAbs by PAGE and protein Bradford assays.
Charge Back Fees and Forms
Dean P. Edwards, Ph.D. (Director)
Dan L. Duncan Cancer Center
Baylor College of Medicine
Margaret M. Alkek Building for Biomedical Research, Room R551E
One Baylor Plaza
Houston, Texas 77030