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Berkeley Structural Genomics Center

Berkeley Structural Genomics Center. NIGMS Protein Structure Initiative. March 7, 2002 Rosalind Kim Didier Busso Jaru Jancarik. NIH Workshop on Protein Production for Structural Genomics. : High-throughput expression screening and approaches to problem proteins Didier Busso.

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Berkeley Structural Genomics Center

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  1. Berkeley Structural Genomics Center NIGMS Protein Structure Initiative March 7, 2002 Rosalind Kim Didier Busso Jaru Jancarik NIH Workshop on Protein Production for Structural Genomics

  2. : High-throughput expression screening and approaches to problem proteins Didier Busso

  3. Clean DNA preparation Clean DNA preparation In vitro screening (using Roche or RIKEN method) Incubation with reaction mix at 30oC (3hrs) or 18oC (overnight) Transformation in expression host Analysis (Dot Blot) Day 1 Growth of starter culture Growth of culture at 30oC (3hrs) and 18oC (overnight) after induction. Harvesting, sonication Day 2 Analysis (SDS-PAGE, Dot Blot) Day 3 Mini-expression Comparison between in vivo and in vitro expression-solubility screening In vivo screening

  4. Comparison In vivo – In vitro Expression In vivo % T O X I C In vitro In vitro system may be used to screen expression of recombinant proteins. In vivo and in vitro Data In vitro Expression Dot Blot I EC731 S I BS843 S I AA967 S I MP865 S I MP004 S I TM678 S I TM172 S I TM142 S 1/3 1/9 1/27 1/81 (dilution)

  5. Purification Selenomethionine Labeled Proteins Nontagged Thermophilic Protein Tagged or Fused Protein +/- Heat Step Heat step Affinity Purification Ion-Exchange Chromatography and/or Size Exclusion Chromatography Ion-Exchange Chromatography SDS/PAGE Dynamic Light Scattering, Mass Spectrometry Crystallization

  6. GGGGGG pHis.MBP(Gly)6 MBP MCS T7 His6 TEV Cloning into pHis.MBP(Gly)6 vector 1) 5 Insoluble proteins: 4 became soluble expressors (10-60mg/L); 1 remained insoluble (30mg/L). 2) 4 Non-expressors: 4 became soluble expressors (5-30mg/L). 3) 3 Low expressors: 3 became high soluble expressors (20-80mg/L). MBP is a good fusion to improve expression and solubility of recombinant proteins. Solubilization by MBP

  7. Aggregated or Insoluble Proteins Insoluble fraction resuspended in buffer with increasing concentrations of salt YES NO Purification Solubilization Matrix * NO Denature protein Use NMR to search for renaturation conditions * Lindwall, G., Chau, M.-F., Gardner, S. R. and Kohlstaedt, L. A. (2000) Protein Engineering13: 67-71. Aggregated or Insoluble Proteins Solubility

  8. (+) The matrix helps to determine the buffer compatible with solubilization. Compatible with automation. GFP is too sensitive as a reporter to correlate activity – solubility. (-) GFP as a Reporter for Solubility Insoluble fraction containing the protein of interest treated by the solubilization matrix. GFP-UV as a reporter. Protein of interest fused at the C-terminus of GFP-UV. C #7 pHisGFP-MP019 10 C, 1 11 20 UV excitation 21 30

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