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The TB Structural Genomics Consortium title

This document outlines the methodologies employed by The TB Structural Genomics Consortium for cloning and protein production at Los Alamos National Laboratory. It details various aspects including vector systems, PCR-based cloning, sample handling schemes, and strategies for optimization of protein expression and solubility. The goal is to screen and produce 400 protein structures, optimizing conditions for recalcitrant proteins and utilizing unique technologies for efficient screening and production workflows. Key processes include GFP-based screening, directed evolution, and innovative protein refolding strategies.

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The TB Structural Genomics Consortium title

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  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.

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