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Heterologous Membrane Protein Expression and Purification from Pyrococcus furiosus

Heterologous Membrane Protein Expression and Purification from Pyrococcus furiosus. Han-Seung Lee , Michi Izumi, Francis E. Jenney Jr, Farris Poole, Frank Sugar, Claudia Shah, Michael W.W. Adams. Southeast Collaboratory for Structural Genomics (SECSG), University of Georgia, .

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Heterologous Membrane Protein Expression and Purification from Pyrococcus furiosus

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  1. Heterologous Membrane Protein Expression and Purification from Pyrococcus furiosus Han-Seung Lee, Michi Izumi, Francis E. Jenney Jr, Farris Poole, Frank Sugar, Claudia Shah, Michael W.W. Adams Southeast Collaboratory for Structural Genomics (SECSG), University of Georgia,

  2. Pyrococcus furiosus • • Optimal growth at 100°C (range 70-105°C) • • Genome is 1.908 Mb and fully sequenced • 2,197 putative single open reading frames (sORFs) • All ORFs were cloned N-terminal His-Tag vector Transmembrane Domain(TMD) prediction programs:PRED-TMR2, TSEG, SOSUI Signal sequence prediction programs: SignalP, SOSUI, TargetP TMDs by PRED-TMR2 1,782 - no TMDs (80%) 452 - TMDs (20%) 139 - 1 TMD (6%) 313 - > 1 TMD (14%) Signal Sequences by SignalP 389 - SS (18%) TMDs + Signal Sequences 271 - both (12%)

  3. Transporter related ORFs in P. furiosus predicted by TransportDB Total transporter related ORFs : 195 ORFs (103 transporters)

  4. Fifty ORFs that did not expressed in E. coli BL21 (DE3) were introduced into C41(DE3) and C43(DE3)

  5. SSE test of membrane associated protein (Project 28) • All selected 50 genes were not expressed in E. coli BL21 (DE3) in SSE or LSE * * * TMD avg = average of TMDs predicted by three different TMD prediction program (TSEG, SOSUI,PRED-TMR2) ** Expression was detected by ELISA

  6. 12 ORFs were selected and expressed in 30 ml culture 12 ORFs were selected and expressed in 30 ml culture 83.3% SDS-PAGE 10 proteins were expressed in 30 ml culture 6 ORFs were selected and fractionated from membrane 33.3% SDS-PAGE 2 proteins were expressed in membrane fraction (Overall success rate ≈ 14%) Mid scale expression test of membrane proteins 50 ORFs were expressed in C41 and C43 50.0% ELISA SSE (Small scale expression, 1 ml culture) 25 proteins showed positive signals in C41 and C43 strains (4 different conditions)

  7. SSE * of membrane proteins (Project 29 & 30) 178 proteins that have 3 TMDs at least were selected and expressed in 30ml scale Project 29 Project 30 * Expression was detected by ELISA 178 membrane proteins  32 proteins were expressed (17%)

  8. Fermentation (5-20L) Sonication Membrane fractionation (Using Triton X-100) Heat treatment (Optional) His-tag purification (Twice – detergent change) Ion-ex chromatography (Optional) Gel filtration Purification procedure for membrane protein

  9. Membrane fractionation Sonication (0.2g wet cell/ml) Centrifuge Supernatant Inclusion body & Undisrupted Ultracentrifuge Supernatant Ppt. (Membrane & IB) Wash membrane pellet 2 x with buffer Ultracentrifuge after each wash Dissolve membrane with buffer containing 1 % Triton-X Stir at 4°C for 3h at least Ultracentrifuge Supernatant Membrane fraction Ppt.

  10. His-tag purification of PF0816 1st purification (Triton X-100) 2nd Purification (OG) SDS-PAGE SDS-PAGE Western blot kDa 177.3 200mM Imidazole 20mM Imidazole 50mM Imidazole 200mM Imidazole Flow-Through 20mM Imidazole 50mM Imidazole 200mM Imidazole Flow-Through Flow-Through 110.7 Marker Marker Marker 79.8 61.0 47.8 35.9 24.5 18.7 13.9

  11. Gel Filtration (Superdex-75) of PF0816 (under 0.8% octyl-β-D-glucopyranoside) SDS-PAGE SDS-PAGE #18 kDa #16 Marker Fraction # 16 177.3 110.7 79.8 61.0 47.8 35.9 concentrated 24.5 18.7 13.9 5.9 Total amount = 1.33mg (From 5 L)

  12. Quality control using MALDI analysis (PF0816) MVALDEPLPY VGVTPMQVLT AIVVLIVGYI VAKVVVASFK RGLKKTKLPE LVVEFLGRFL SALLYVAVIL LAVRALGIEV GSVVLGLSAV IGLILGFGMQ DTLTNLAAGV WIAALRPIDI GEVVEVAGKV GKVNAVGIMS TELLTADNVL ITIPNKLVWG NVITNYTRMP TRRVDVNVGV AYGTDLDKAI KVAMELMQNH PKVLKDPAPAVVVTELGDSSINLQLRAWAK TEDYWTVKFD LTKGIYEAYR REGIEIPFPQ LDVHIKEMPK Underlined sequence : Predicted TMDs Red sequence : Peptides from MALDI Mw Mw in DB Difference A. acid sequence 871.2820 871.4314 -0.1494 244 250 (K) GIYEAYR(R) 1435.5930 1435.7698 -0.1768 157 168 (K) LVWGNVITNYTR(M) 1734.6980 1734.9430 -0.2450 252 266 (R) EGIEIPFPQLDVHIK(E) 2193.9040 2194.1719 -0.2679 206 226 (K) DPAPAVVVTELGDSSINLQLR(A) 2534.1320 2534.4194 -0.2874 203 226 (K) VLKDPAPAVVVTELGDSSINLQLR(A) 0 unmatched masses: The matched peptides cover 21% (58/270 AA's) of the protein. Coverage Map for This Hit (MS-Digest index #): 887

  13. Crystals of PF0816 The protein crystallized in 12 % PEG 4000 100 mM NaCl 100 mM Lithium Sulfate 100 mM HEPES (pH 7.6)

  14. Conclusions • C41(DE3) and C43(DE3) are useful host for membrane protein expression and C43(DE3) is better than C41(DE3). • Membrane proteins were purified using the “crystallography-favored” detergent, octyl-β-D-glucopyranoside (OG). • 3. If membrane proteins were sufficiently expressed in membrane, three can be purified within a week. • Work in progress • 1. Membrane proteins with signal peptide •  expression in C-terminal His-tag vector • 2. Optimization of expression condition •  Autoinduction media and optimization of fermentation condition • 3. Membrane protein complex •  Coexpression of membrane protein complex

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