1 / 10

The TB Structural Genomics Consortium title

The TB Structural Genomics Consortium title. Cloning Platform At Los Alamos National Laboratory. NIGMS, Protein Production Workshop, 3/8/02. General System. Vector: N-6-his-GFPsf (pET21/28 hybrid vector) Insert: PCR, double cohesive ends (NdeI/BamHI, NdeI/SpeI) 96 sample handling scheme

faith-hood
Download Presentation

The TB Structural Genomics Consortium title

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. The TB Structural Genomics Consortiumtitle Cloning Platform At Los Alamos National Laboratory NIGMS, Protein Production Workshop, 3/8/02

  2. General System • Vector: N-6-his-GFPsf (pET21/28 hybrid vector) • Insert: PCR, double cohesive ends (NdeI/BamHI, NdeI/SpeI) • 96 sample handling scheme • No complete automation system • Modules of parallel sample handling system • 96 vacuum filter system • 96-pipette hydra • 96-well plate sonicator • SDS-PAGE system for 96 sample analysis

  3. General scheme? • General strategy for 96 clone production • PCR reaction: 8 channel pipetts • PCR amplicon clean up: 96 vacuum filter • Amplicon/insert analysis: 96 sample system • Ligation and transformation: PCR machine • Transformation/Plating: 96 zone grid system • Colony picking: GFP based screening • Expression/solubility/binding assay: • plate sonicator, bead assay

  4. Strategy to meet Consortium Goal of 400 structures • Screen 1500 ORFs • - 300 suitable for production • Process recalcitrant proteins • - 700 refold or evolve for improved solubility • - 500 poorly expressed one to be • evolved for expression

  5. Strategies to cope with insoluble proteins • Partial factorial protein refolding screens • Armstrong et al., (Protein Science 8, 1475, 1999) • Directed Evolution using protein folding • And solubility reporters • Waldo et al., (Nature Biotechnology 17, 691, 1999) • Kim, C. A. et al., (EMBO J. 20, 4173, 2001)

  6. Unique Technologies that are ready to be Transferred (I) • Inducible serine-suppressor vector eliminating sub-cloning for GFP-free target protein • Super-folder GFP fusion for identifying expressers • GFP-based directed evolution protocol for engineering soluble variants

  7. Unique Technologies that are ready to be Transferred (II) • Small scale IMAC binding assay protocol to identify candidates for a large scale protein production

  8. Terrific Broth Improves Cell Density and Protein Yield Rv2965C LB medium Rv2965C TB medium IT: Induction time (hours)

  9. GFP is useful for monitoring sonication efficiency

  10. GFP Pellet Supernatant S5 and S10: Sonication for 5 and 10 minutes Fig5. Electrophoretic patterns of samples after cell disruption by CelLytic B. Rv3110 BugBuster (Novagen) CelLytic B (Sigma) T:Total, S:Soluble, I:Insoluble, F:Flow-through, W:Wash, E:Elution, R: Regeneration Fig6. Electrophoretic patterns of each fraction of IMAC.

More Related