Protein Expression Research Group (PERG) 2012 Study
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1 Protein Expression Research Group (PERG) 2012 Study Richard J Heath 1, Fei P Gao 2, Pamela Scott Adams 3, Cynthia Kinsland 4, James Bryson 5, Bo Xu 6, Thomas Neubert 7. 1 St Jude Children's Research Hospital, 2 University of Kansas, 3 Trudeau Institute, 4 Cornell University, 5 Bristol- Myers Squibb Co., 6 University of Texas Medical Branch, 7 New York University
2 Insoluble Proteins in E. coli Many overexpressed proteins in E. coli (up to 50%) form insoluble inclusion bodies Inclusion bodies contain primarily unfolded ectopic protein If biologically ac3ve protein is required, inclusion bodies are o9en deemed undesirable However, they can be a ready source of rela3vely pure protein
3 The PERG 2012 Study Goal: To get protein labs to consider refolding proteins as a viable op3on Outline: Protein that expresses as inclusion body Simple, validated methods for extrac3on, solubiliza3on and refolding Simple assay for refolded protein
4 General Methods for Protein Refolding Chromatographic Solvent exchange by size exclusion On column refolding Immobilized chaperone assisted refolding Non Chromatographic Rapid Dilu3on Dialysis
5 Structure of ATP Synthase γ Subunit
6
7 The Chloroplast ATP Synthase γ-subunit-gfp Fusion Protein where GFP was inserted
8 Sequence of the Chloroplast ATP Synthase γ-subunit-gfp Fusion Protein ANLRELRDRIGSVKNTQKITEAMKLVAAAKVRRAQEAVVNGRPFS ETLVEVLYNMNEQLQTEDVDVPLTKIRTVKKVALMVVTGDRGLCG GFNNMLLKKAESRIAELKKLGVDYTIISIGKKGNTYFIRRPEIPVDRY FDGTNLPTAKEAQAIADDVFSLFVSEEVDKVEMLYTKFVSLVKSDP VIHTLLPLSPKGEICDINGKCVDAAEDELFRLTTKEGKSKGEELFTGV VPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPW PTLVTTFAYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKD DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSH NVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGP VLLPDNHYLSTQSALSKDPWSHPQFEKRDHMVLLEFVTAAGITHG MDELYKLTVERDMIKTETPAFSPILEFEQDPAQILDALLPLYLNSQIL RALQESLASELAARMTAMSNATDNANELKKTLSINYNRARQAKIT GEILEIVAGANSCV
9 Protocol for expression and inclusion body isola3on Express γ ATP synthase GFP fusion protein in BL21(DE3) LB medium, 3 hr 37 C Harvest cell pellet Isolate and solubilize inclusion bodies Suspend cells in Tris buffer and sonicate briefly Centrifuge and resuspend pellet in Tris buffer Repeat 3 3mes
10 Protocol for solubiliza3on and refolding Solubilize and refold the fusion protein Resuspend pellet in Tris buffer with 8M urea or 6 M guanidine HCl Centrifuge to remove debris Dialyze against 500 vols of Buffer 1 (4 M urea) Dialyze against 500 vols of Buffer 2 (2 M urea) Centrifuge to remove debris. Protein is now in solu3on (which should be green!) Measure absorbance & fluorescence of refolded protein Major absorp3on peak at a wavelength of 395 nm and a minor one at 475 nm Excita3on wavelength 395 nm with fluorescence emission peak is at 509 nm
11 Fluorescence Emission Spectrum of Refolded γ-atps-gfp Fusion Protein
12 Fluorescence Spectrum of MBCF γ- ATPS-GFP Fusion Protein 395 Excitation/Absorbance peak No emission peak?
13 EGFP [synthetic construct]
14 EGFP runs as a dimer and fluoresces after refolding B A B A Coomassie Fluorescence
15 Revised folding study Provided by P Gao Folded protein EGFP Standard Inclusion body prep Plasmid DNA for 6 His-TEV-EGFP
16 Protocol for solubilization and refolding Solubilize and refold the fusion protein Resuspend pellet in Tris buffer with 8M urea at ~1 mg/ml Centrifuge to remove debris Dialyze against 500 vols of Buffer 1 (4 M urea) Dialyze against 500 vols of Buffer 2 (2 M urea) Centrifuge to remove debris. Measure absorbance & fluorescence of refolded protein Major absorption peak at a wavelength of 488 nm Excitation wavelength 488 nm with fluorescence emission peak at 509 nm
17 Fluorescence Spectrum of MBCF GFP vs Std EGFP Std EGFP MBCF EGFP Excitation/Absorbance peak Emission Peak Excitation/Absorbance peak Emission Peak Very green Slight green tinge,but no emission peak? CONCENTRATE!
18 Fluorescence Emission Spectrum of Refolded MBCF EGFP vs Std EGFP Std EGFP Std EGFP MBCF EGFP MBCF EGFP
19 Conclusions Synthetic EGFP construct is a good tool for a folding study. Should be foldable by non expert protein personnel. Participants can participate a 2 different levels Folding only Protein Expression and folding
20 What Next? Ask Phillip Gao for the plasmid! Send a copy of you absorbance and/or fluorescent spectrum back once you have refolded the protein Results to be presented next year
21 One-step Batch Affinity Chromatography: Protein Refolding for Dummies Major equipment: one column, two hands. Method: Incubate with Ni2+ resin. Wash / refold: on column, gravita3on flow. Elute protein. Collect, especially the front frac3on. SL FT wash Elution 12% SDS PAGE of Rv0008c, a membrane protein from TB
22 When the refolding work: Lesson 1: Protein production in NOT the bottleneck for Membrane Protein Structural Genomics. 18 TB membrane protein spectra were obtained of which more than 90% of the expected amide 15 N to 1 H correlation resonances were observed. 2 were completely assigned.
23 Protein Expression Research Group (PERG): We focus on recombinant protein expression and purifica3on Help needed for upcoming events: 1. Protein refolding study: participation. 2. Recruiting new members: recommendation. 3. Organizing a workshop on production of recombinant protein: input.
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