Current Antigen Production Effort
Common Fund FOA1: Production Of Human Proteins To Be Used For Generating Affinity Reagents (U01) RFA-RM-10-007.
Stephen Anderson, Ph.D.
Center for Advanced Biotechnology & Medicine (CABM)
Department of Molecular Biology & Biochemistry
tel. 732-235-5022 (direct)
Bioinformatics analyses of human transcription factor (hTF) sequences will be used to identify domains that can fold into defined three-dimensional structures bearing specific 3D epitopes. Human TF target domains will be expressed using a variety of expression systems and tags. All targets will be expressed as fusions to at least two tags: (a) a His tag for preliminary purification by immobilized metal affinity chromatography, and (b) a secondary tag [currently biotin-mimetic peptides] to enable a “polishing” affinity purification step and/or immobilization to a solid support; additionally, a TEV protease cleavage site will be included to allow tag removal via proteolysis. Initially, all targets will be screened for expressability on a small scale using pET-based E. coli expression systems. For more difficult targets that are refractory to expression in E. coli, several eukaryote-based expression systems will be employed: P. pastoris, baculovirus/insect cells, wheat germ cell free systems, and human HEK293 cells. All immunogens will be characterized biophysically, including analyses by electrospray ionization-mass spectrometry, and tested for endotoxin prior to handoff of samples to the production awardees. The plan is to deliver purified and characterized hTF immunogens at the 1 - 2 mg scale to the affinity reagent producing partner(s) on an ongoing basis. Samples will be sent to the affinity reagent producing group(s) (RFA-RM-10-17) according to a mutually agreed upon schedule, and shipping will commence as soon as each production partner is identified and ready to receive them. In parallel with shipping the protein samples to the affinity reagent producing partner(s), plasmid samples will be sent for archiving and public distribution to the DNASU Plasmid Repository at Arizona State University. Potential applicants responsive to RFA-RM-10-018 should include material shipping/handling costs if requesting antigen reagents supported by this RFA-RM-10-007.