Didier Busso by dandanhuanghuang


									                                      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
                                             Protein Production Workshop
Berkeley Structural Genomics Center


         High-throughput expression screening and approaches
                         to problem proteins

                                      Didier Busso

                                      Protein Production Workshop
Berkeley Structural Genomics Center

           Comparison between in vivo and in vitro expression-solubility screening
                       In vivo screening                                        In vitro screening
                                                                           (using Roche or RIKEN method)

                     Clean DNA preparation                                     Clean DNA preparation

             Transformation in expression host                      Incubation with reaction mix at 30oC (3hrs)
                                                                               or 18oC (overnight)

                                                          Day 1                 Analysis (Dot Blot)
                    Growth of starter culture

    Growth of culture at 30oC (3hrs) and 18oC              Day 2
 (overnight) after induction. Harvesting, sonication

              Analysis (SDS-PAGE, Dot Blot)                Day 3

                                                 Protein Production Workshop
                                           In vivo and in vitro Data
Berkeley Structural Genomics Center

                     In vitro Expression                     Comparison In vivo – In vitro Expression
                           Dot Blot

                 I                             100
  EC731          S
  BS843          S                              80
  AA967          S
                 I                                                                                        Sol.
                                                60                                                               In vivo
  MP865                                                                                                   Ins
                 S                             %
                 I                                                         T                              Sol.
                                                                                                                 In vitro
  MP004                                         40
                                                                           O                              Ins.
                 I                                                         X
  TM678          S                                                         I
                 I                                                         C
  TM142                                                 EC731 BS843 AA967 MP865 MP004 TM678 TM172 TM142
                        1/3    1/9 1/27 1/81

     In vitro system may be used to screen expression of recombinant proteins.
                                                       Protein Production Workshop
Berkeley Structural Genomics Center

                                        Selenomethionine Labeled Proteins

                                      Nontagged Thermophilic Protein         Tagged or Fused Protein
                                                                                         +/- Heat Step
                                                      Heat step
                                                                               Affinity Purification
                                      Ion-Exchange Chromatography
                                                                          Ion-Exchange Chromatography
                                      Size Exclusion Chromatography

                                              Dynamic Light Scattering, Mass Spectrometry

                                                       Protein Production Workshop
                                              Solubilization by MBP
Berkeley Structural Genomics Center


       pHis.MBP(Gly)6                 T7                    MBP                       MCS

                                       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.

                                                 Protein Production Workshop
                                      Aggregated or Insoluble Proteins
Berkeley Structural Genomics Center

                                                   Aggregated or Insoluble Proteins

                          Insoluble fraction resuspended in buffer with increasing concentrations of salt


                                               NO                                               YES

                  Solubilization Matrix *                                                                 Purification


             Denature protein
Use NMR to search for renaturation conditions

                                      * Lindwall, G., Chau, M.-F., Gardner, S. R. and Kohlstaedt, L. A. (2000) Protein Engineering 13: 67-71.

                                                         Protein Production Workshop
                                      GFP as a Reporter for Solubility
Berkeley Structural Genomics Center

      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

                                                                                     C, 1       10

                       UV excitation                                                 11         20

                                                                                     21         30

           (+) The matrix helps to determine the buffer compatible with solubilization.
               Compatible with automation.
           (-) GFP is too sensitive as a reporter to correlate activity – solubility.

                                              Protein Production Workshop

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