dhvi protein production facility
TRANSCRIPT
DHVI Protein Production Facility
Technology / Instruments / Resources Mission
Services
• The Protein Production Facility (PPF) was established with a grant awarded by the Bill & Melinda Gates Foundation with the mission of providing high-quality, research-grade recombinant proteins and antibodies to the members of the Collaboration for Aids Vaccine Discovery (CAVD). In July 2017, the PPF opened its doors as a DHVI Shared Resource Facility that is open to all Duke University researchers.• The Mission of the DHVI PPF is to provide high-quality proteins and
antibodies for in vitro assays for basic research and clinical study immune monitoring and to facilitate immunogen discovery.• The production of all proteins and antibodies is governed by a Quality
Management plan with oversight by the Quality Assurance for Duke Vaccine Immunogenicity Program (QADVIP), Duke’s Quality Assurance Unit. All proteins and antibodies are produced following Standard Operating Procedures (SOPs) and using Good Documentation Practices. Following production, proteins and antibodies undergo a quality control panel and are visualized by SDS-PAGE under reduced and non-reduced conditions and tested for sterility, mycoplasma and endotoxin.
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• Protein Production (1L and 4L Transfections in 293 cells or CHO cells)• Antibody/Fab Production (1L and 4L Transfections in 293 cells)• FPLC/SEC• Endotoxin Testing (individual samples) • Consulting for antibody and HIV-1 Env sequence modifications/design
• Protein expression and available nickel, lectin, protein A, G or M, and Kappa select purification methods
• Recombinant Viral Envelope Proteins, SOSIP trimers and Fc Receptors
• Recombinant Immunoglobulin (Ab or Fab)• Endotoxin testing• FPLC/SEC
Recent Projects / Publications
Reservations and Service Requests
To submit a service request, go to CoreResearch@Duke and select the DHVI Protein Production Facility (D-0078).
• PPF has made over 200 different viral envelope proteins and 60 different Antibodies• Production of 4 CD4-IgG variants for Michael Farzan at Scripps through a
CAVD collaboration (Total production was > 12 grams)• Production of HC1 HC1/SAP10, CH505.TF 6R.SOSIP.664 and
BG505.6R.SOSIP.664trimersRepresentative Publications Supported by DHVI PPF :• Shen X, Basu R, Sawant S, Beaumont D, Kwa SF, LaBranche C, Seaton
KE, Yates NL, Montefiori DC, Ferrari G, Wyatt LS, Moss B, Alam SM, Haynes BF, Tomaras GD, Robinson HL. 2017. HIV-1 gp120 and modified vaccinia virus Ankara (MVA) gp140 boost immunogens increase immunogenicity of a DNA/MVA HIV-1 vaccine. J Virol 91:e01077-17.• Townsley S, Mohamed Z, Guo W, McKenna J, Cleveland B, LaBranche
C, Beaumont D, Shen X, Yates NL, Pinter A, Tomaras GD, Ferrari G, Montefiori DC, Hu SL. Induction of Heterologous Tier 2 HIV-1-Neutralizing and Cross-Reactive V1/V2-Specific Antibodies in Rabbits by Prime-Boost Immunization. J Virol. 2016 Sep 12;90(19):8644-60. doi: 10.1128/JVI.00853-16. Print 2016 Oct 1. PubMed PMID: 27440894; PubMed Central PMCID: PMC5021413.
Leadership / Experience • Frederick Porter, PhD, Sr. Director of Product Development at DHVI,
brings industrial expertise in process management and antigen manufacturing• James Peacock, PhD, PPF Director, has 5 years experience managing
the production of antibodies and HIV1-Envs• Cynthia Nagle, PhD, PPF Program/Project Manager• Kathy Yarborough, MS, PPF Laboratory Manager• Kevin Saunders, PhD, Scientific Advisor, Antibody, HIV-1 ENV/SOSIP
trimer design
Contact Us
Process Through the PPF
SOPs and Quality Management Plan Govern PPF Processes
Work Flow for Protein Production
QC Tests Performed Sterility- tested for aerobic and anaerobic bacteria Mycoplasma- qualitative test for all common mollicute contaminations Endotoxin- quantitative, kinetic assay for the detection of endotoxin SDS-PAGE and Western Blot Sequence verification by LC/MS
Plasmids with the insert produced and purified
PurificationIncubated shaking 4 days
in 8% CO2 at 37°C.
Protein aliquots stored - 80°C until shipped.
Transiently transfect 293 Supernatants
harvested and clarified
Eluted protein is buffer exchanged into PBS, filtered and aliquots made
QC release testing performed prior to delivery
Endotoxin Assay
If you are interested in collaborating with the Protein Production Facility, please contact: [email protected]
Cynthia Nagle, PhD Program Manager919-660-0352 [email protected]
Jamie Peacock, PhD Protein Production Facility Director919-613-6149 [email protected]
CO2 Incubators and Biosafety Cabinets for Project Segregation
QC Equipment AKTA pure with Fraction Collector