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    1. Cloning, Expression, Purification and Enzymological Characterization of NS2B/NS3 Protease / RNA Helicase protein of Japanese Encephalitis Virus. Chakard Chalayut Advisor: Asst. Prof. Gerd Katzenmeier, Ph.D. Laboratory of Molecular Virology Institute of Molecular Biology & Genetics
    2. Japanese Encephalitis Virus (JEV) -Flaviviridae family -Mosquito-borne neurotropic flavivirus •causes severe central nerve system diseases
    3. Japanese Encephalitis Virus (JEV) Culex tritaeniorhynchus. Source: fehd.gov.hk
    4. Japanese Encephalitis Virus (JEV)
    5. Japanese Encephalitis Virus (JEV) Source : vietnammedicalpractice.com
    6. Japanese Encephalitis Virus (JEV)
    7. Japanese Encephalitis Virus (JEV) JEV causes severe central nerve system diseases such as p o l i o mye l i t i s - l i ke acute flaccid paralysis, aseptic meningitis and encephalitis Source:wonder.cdc.gov
    8. Japanese Encephalitis Virus (JEV)
    9. Japanese Encephalitis Virus (JEV)
    10. Japanese Encephalitis Virus (JEV) 50,000 Cases
    11. Japanese Encephalitis Virus (JEV) 30% fatality rate
    12. Japanese Encephalitis Virus (JEV) 10,000 Cases
    13. Prevention and treatment of JEV disease
    14. Prevention and treatment of JEV disease Drug No drug exist
    15. Prevention and treatment of JEV disease Drug No drug exist Vaccine development Available vaccine
    16. Prevention and treatment of JEV disease Drug No drug exist Vaccine development Available vaccine Elimination of mosquitoes Mosquitoes control breeding places
    17. Molecular biology of Japanese Encephalitis Virus
    18. The NS2B • 130 aa • activating domain central hydrophilic region (Falgout et al, 1993) • 3 membrane spanning parts
    19. The NS2B • 130 aa • activating domain central hydrophilic region (Falgout et al, 1993) • 3 membrane spanning parts hydrophobicity plot
    20. The NS2B • 130 aa • activating domain central hydrophilic region (Falgout et al, 1993) • 3 membrane spanning parts hydrophobicity plot 51 DMWLERAADISWEMDAAITGSSRRLDVKLDDDGDFHLIDDPGVP 95
    21. The NS2B • 130 aa Hypothetical model • activating domain NS2B-NS3 complex central hydrophilic region (Falgout et al, 1993) • 3 membrane spanning parts hydrophobicity plot ฺBrinkworth et al, 1999 51 DMWLERAADISWEMDAAITGSSRRLDVKLDDDGDFHLIDDPGVP 95
    22. The NS3 Theoretical model from PDB 2I84
    23. The NS3 Protease Theoretical model from PDB 2I84
    24. The NS3 Protease NTPase RNA Helicase Theoretical model from PDB 2I84
    25. The NS3 •Chymotrypsin-like fold 2-β barrel domains •Inactive alone Theoretical model from PDB •Enzyme’s pocket is small 2I84
    26. The NS3 protease
    27. The NS3 protease •NS3 serine protease domain 20 kDa •catalytic residues His51, Asp75, Ser135
    28. Background • Lin. C W et al,2007
    29. Background • Ser to Ile60 were essential region required for NS3 46 protease activity. • Ala substition of Trp 50, Glu55, and Arg56 in NS2B shown significantly reduced NS3 protease activity. • Lin. C W et al,2007
    30. Objective
    31. Objective • to perform cloning of the NS2B-NS3 portion of the JEV polyprotein, express in E.coli and biochemically purify to determinants of clevage activity and cofactor requirement will be analyzed and compared to dengue virus. • The second objective is to study differences in substrate specificity and inhibitors by using peptide substrates incorporated with fluorogenic or chromogenic reported groups.
    32. Method & Result pLS with NS2B-NS3 JEV
    33. Method & Result pLS with NS2B-NS3 JEV NS2B(H) NS3p
    34. Method & Result pLS with NS2B-NS3 JEV NS2B(H) NS3p SOE-PCR
    35. Method & Result pLS with NS2B-NS3 JEV NS2B(H) NS3p SOE-PCR NS2B(H)-NS3p
    36. NS2B(H) NS2B(H) Control NS3 protease NS3 protease NS3 protease Control 1500 1500 1000 900 800 1000 700 900 800 600 700 500 600 400 500 400 300 300 200 200 Figure 1 : The PCR product NS2B(H) JEV amplified from NS2B-NS3 Figure 2 : The PCR product NS3protease JEV amplified from NS2B- JEV (Lane 3 and 4). The size of NS2B(H) was 187 bp. NS3 JEV (Lane 3 to 5 ). The size of NS3 protease was 594 bp.
    37. Control NS2B(H)-NS3p NS2B(H)-NS3p Control 1500 1000 900 1500 800 700 600 1000 900 500 800 400 700 600 300 500 400 200 300 100 Figure 4 : The NS2B(H)-NS3protease JEV (Lane 3 ) after digested with BamHI and KpnI. The size of NS2B(H)-NS3 protease was 765 bp. Figure 3 : The SOE-PCR product NS2B(H)-NS3protease JEV (Lane 2 to 5 ). The size of NS2B(H)-NS3 protease was 765 bp.
    38. Method & Result
    39. Method & Result NS2B(H)-NS3p JEV pTrcHis A Ligation & Transformation Site Screening & Digest with Restriction Enzyme
    40. 23.13 kb 2.03 kb 2.32 kb 4.56 kb 6.56 kb 9.42 kb pTrc plasmid Control Size Screening Clone 1 Clone 2 Clone 3 Clone 4 Clone 5 Clone 6 Clone 7 Clone 8 Clone 9 Clone 10 Clone 11 Clone 12 Clone 13 Clone 14 Clone 15 Clone 16 Clone 17
    41. Digest with Restriction Enzyme clone 1 undigested clone 1 with BamHI,KpnI clone 1 with BamHI • Lane 1 : λ/BstII marker •Lane 2 : Clone 1 with BamHI and KpnI digerstion 8.454 kb 7.242 kb •Lane 3 : Clone 1 with BamHI digestion 6.369 kb 5.686 kb 4.822 kb 4.324 kb •Lane 4 : Clone 1 without digestion 3.645 kb 2.323 kb 1.929 kb 1.371 kb 1.264 kb 702 bp
    42. Sequencing of candidate clone.
    43. Deduced amino acid sequences of Japanese encephalitis virus NS2B(H) NS2B(H) C-terminal NS3p
    44. Expression of Japanese encephalitis virus NS2B(H)/NS3 protease at different time. 0 Hr 1% 2 Hr 4 Hr 6 Hr incubate overnight O.D.= 0.4-0.6 8 Hr induction by IPTG
    45. SDS-PAGE Analysis of Japanese encephalitis virus NS2B(H)/NS3 protease at different time. marker ptrc 0- 8 Hr. NS2B(H)/NS3p 0- 8 Hr. 210 kD 125 kD 101 kD 56.2 kD 35.8 kD NS2B(H)/NS3 29 kD NS3 21 kD 6.9 kD NS2B(H)
    46. Western blot analysis of Japanese encephalitis virus NS2B(H)/NS3 protease at different time. marker NS2B(H)/NS3p 0- 8 Hr. 35.8 kD NS2B(H)/NS3 protease 29 kD 21 kD NS3 NS2B(H)
    47. Expression of Japanese encephalitis virus NS2B(H)/NS3 protease expressed at different temperatures. 1% 18 ˚C 25 ˚C 37 ˚C incubate overnight O.D.= 0.4-0.6 induction by IPTG Incubate 8 Hrs.
    48. SDS-PAGE analysis of Japanese encephalitis virus NS2B(H)/NS3 protease expressed at different temperatures. pTrc 0 hr 37 ˚C JEV 37 ˚C JEV 18 ˚C JEV 30 ˚C pTrc 18 ˚C pTrc 25 ˚C pTrc 37 ˚C pTrc 30 ˚C JEV 25 ˚C marker 210 kD 125 kD 101 kD 56.2 kD 35.8 kD NS2B(H)/NS3 29 kD 21 kD NS3 protease 6.9 kD NS2B(H)
    49. Western blot analysis of Japanese encephalitis virus NS2B(H)/NS3 protease expressed at different temperatures. NS2B(H)/NS3p 37 ˚C NS2B(H)/NS3p 18 ˚C NS2B(H)/NS3p 30 ˚C NS2B(H)/NS3p 25 ˚C pTrc 0 hr 37 ˚C pTrc 18 ˚C pTrc 25 ˚C pTrc 37 ˚C pTrc 30 ˚C marker 101 kD 35.8 kD NS2B(H)/NS3 NS3 protease 21 kD NS2B(H) 6.9 kD
    50. Expression of Japanese encephalitis virus NS2B(H)/NS3 protease expressed at different IPTG concentrations 0.1 mM 0.2 mM 1% 0.3 mM 0.4 mM 0.5 mM incubate overnight 0.6 mM 0.7 mM O.D.= 0.4-0.6 0.8 mM induction by IPTG Incubate 8 Hrs.
    51. SDS-PAGE analysis of Japanese encephalitis virus NS2B(H)/NS3 protease expressed at different IPTG concentrations. 0.1mM 0.8mM marker 210 kD 125 kD 101 kD 56.2 kD 35.8 kD NS2B(H)/NS3 29 kD NS3 protease 21 kD 6.9 kD NS2B(H)
    52. Western blot analysis of Japaneseencephalitis virus NS2B(H)/NS3 protease expressed at different IPTG concentrations 0.1mM 0.8mM marker 35.8 kD NS2B(H)/NS3 protease 29 kD
    53. Expression of Japanese encephalitis virus NS2B(H)/NS3 protease at small scale expression.
    54. Expression of Japanese encephalitis virus NS2B(H)/NS3 protease at small scale expression. 1% Centrifuge, Lysis, Sonication and Collect incubate overnight fraction O.D.= 0.4-0.6 induction by o.1 mM IPTG Incubate 8 Hrs.
    55. SDS-PAGE analysis of Japanese encephalitis virus NS2B(H)/NS3 protease at small scale expression. marker C T I S 210 kD 125 kD 101 kD Lane 1: maker 56.2 kD NS2B(H)/NS3 protease Lane 2: Control pTrcHis Lane 3: Total fraction (T) 35.8 kD Lane 4: Insoluble fraction (I) Lane 5: Soluble fraction (S) 29 kD NS3 protease 21 kD NS2B(H) 6.9 kD
    56. Western blot analysis of Japanese encephalitis virus NS2B(H)/NS3 protease at small scale expression. T I S 35.8 kD NS2B(H)/NS3 protease Lane 1: maker Lane 2: Control pTrcHis Lane 3: Total fraction (T) Lane 4: Insoluble fraction (I) Lane 5: Soluble fraction (S)
    57. SDS-PAGE analysis of Japanese encephalitis virus NS2B(H)/NS3 protease at large scale expression. marker marker T I S TI S 210 kD 101 kD 125 kD 56.2 kD Lane 1: maker Lane 2: Total fraction (T) 35.8 kD NS2B(H)/NS3 protease Lane 3: Insoluble fraction (I) 29 kD Lane 4: Soluble fraction (S) NS3 protease 21 kD 6.9 kD NS2B(H)
    58. Western blot analysis of Japanese encephalitis virus NS2B(H)/NS3 protease at large scale expression. marker marker TI S T I S Lane 1: maker NS2B(H)/NS3 protease Lane 2: Total fraction (T) Lane 3: Insoluble fraction (I) Lane 4: Soluble fraction (S) NS3 protease NS2B(H)
    59. Purification of the NS2B(H)/NS3p protein harboring the N-terminal polyhistidine tag The NS2B(H)/NS3 protease was purify by HiTrap Chelating column.
    60. Method Soluble fraction in buffer H Wash with Buffer H (30 mM imidazole) Elute with Buffer H (100 mM imidazole)
    61. SDS-PAGE analysis of Japanese encephalitis virus NS2B(H)/NS3 protease from Hi-trap purification. Elute and concentrate Washing fraction Soluble fraction fraction Flowtrough unduction induction marker 210 kD 125 kD 101 kD 56.2 kD NS2B(H)/NS3 protease 35.8 kD 29 kD NS3 protease 21 kD 6.9 kD
    62. Western-blot analysis of Japanese encephalitis virus NS2B(H)/NS3 protease from Hi-trap column purification Elute and concentrate Washing fraction Soluble fraction fraction unduction Flowtrough induction marker NS2B(H)/NS3 35.8 kD 29 kD NS3 protease 21 kD NS2B(H) 6.9 kD
    63. Characterize the activity of NS2B(H)/ NS3p JEV with Ac-RRRR-pNA The substrate Ac-RRRR-pNA is based on the para- nitroanilide principle the enzyme will cleave between the tetra arginine and release pNA Free pNA will be monitored at A405 by the spectrophotometry method. The change of pNA will change the color of buffer and correlate to the activity of the enzyme
    64. Result Trypsin NS2B(H)/NS3p JEV substrate NS2B(H)/NS3p JEV = 10 μM trypsin = 0.4 μM substrate = 500 μM
    65. Problem • The protein has by products from the purification step. • No activity • The fusion protein was different from the previous work of JEV.
    66. The International Journal of Biochemistry & Cell Biology 39 (2007) 606–614
    67. Method Soluble fraction in buffer A (50mM Hepes pH 7.0, 500mM NaCl) Wash with Buffer A (30 mM imidazole) Elute with Buffer A (100 mM imidazole)
    68. Method The eluted protein (superdex 75 HR 10/300)
    69. peak1 peak2
    70. 101 kD 210 kD 125 kD 6.9 kD 29 kD 21 kD 56.2 kD 35.8 kD Soluble Flowthrough wash Elute peak 1 peak2 result Soluble Flowthrough wash Elute peak 1 peak2
    71. 29 kD 21 kD 101 kD 210 kD 6.9 kD 125 kD 56.2 kD 35.8 kD uninduction induction Soluble Flowthrough wash Elute Gel filtration
    72. 29 kD 21 kD 101 kD 210 kD 6.9 kD 125 kD 56.2 kD 35.8 kD uninduction induction Soluble Flowthrough wash Elute Gel filtration
    73. 29 kD 21 kD 101 kD 210 kD 6.9 kD 125 kD 56.2 kD 35.8 kD induction Soluble Flowthrough wash Elute Gel filtration
    74. 29 kD 21 kD 101 kD 210 kD 6.9 kD 125 kD 56.2 kD 35.8 kD induction Soluble Flowthrough wash Elute Gel filtration
    75. The substrate
    76. The substrate
    77. Protein assay method 7-Amino-4-Methyl Coumarin (AMC) standard curve • A serial dilution of AMC, from 3 μM - 50 μM, was prepared in 100 μl assay buffer (200 mM Tris-HCl pH. 9.5, 13.5 mM NaCl and 30% Glycerol). • incubated at 37 ํC for 30 minutes • Fluorescence signals were measured at an excitation wavelength 355 nm and emission 460 at 37 ํC nm. Fluorescence signals was plotted against AMC concentration
    78. result Data 1 100000 Fluorescence unit 80000 Fluorecence units 60000 40000 20000 0 0 20 40 60 [AMC] (µM) The correlation between units with AMC concentrations was calculated from 1/ slope of linear regression. 1 fluorescence unit = 6.839 nM [AMC]
    79. Protein assay method Enzyme Activity assay NS2B(H)-NS3p JEV protein 100 μl assay buffer (200 mM Tris-HCl pH. 9.5, 13.5 mM NaCl and 30% Glycerol) • incubated at 37 ํC for 30 minutes adding 200 μM of Pyr-RTKR-AMC substrate • Fluorescence signals were measured at an excitation wavelength 355 nm and emission 460 at 37 ํC nm and monitored every 3 minutes for 2 hr.
    80. result Data 1 30000 Control 200 mM Fluorescence unit 20000 10000 0 0 50 100 150 time Progress curves for enzymes-catalyzed hydrolysis observed at 200 μM of Pyr-RTKR-AMC were plotted between fluorescence signals against time
    81. Protein assay method Determination of kinetic parameters NS2B(H)-NS3p JEV protein 100 μl assay buffer (200 mM Tris-HCl pH. 9.5, 13.5 mM NaCl and 30% Glycerol) • incubated at 37 ํC for 30 minutes Pyr-RTKR-AMC was varied the concentrations from 15 μM to 500 μM • Fluorescence signals were measured at an excitation wavelength 355 nm and emission 460 at 37 ํC nm and monitored for 2 hr.
    82. result Data 1 2000 1500 Velocity (nM/min) 1000 500 0 0 200 400 600 [Pyr-RTKR-AMC] (µM) Vmax (nM/min) Km (µM) Kcat (s-1) Kcat/ Km(M-1s-1) 2672 226.4 3.22 0.014
    83. What’s next? • Try to improve purification and find the amount of active protein • compare to Den NS2B(H)-NS3p

    + Chakard ChalayutChakard Chalayut, 2 years ago

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