Cloning, Expression, Purification and
 Enzymological Characterization of
 NS2B/NS3 Protease / RNA Helicase
             pro...
Japanese Encephalitis Virus
              (JEV)

-Flaviviridae family
-Mosquito-borne neurotropic flavivirus


          •c...
Japanese Encephalitis Virus
          (JEV)



  Culex tritaeniorhynchus.




                             Source: fehd.go...
Japanese Encephalitis Virus
          (JEV)
Japanese Encephalitis Virus
          (JEV)




  Source : vietnammedicalpractice.com
Japanese Encephalitis Virus
          (JEV)
Japanese Encephalitis Virus
                  (JEV)
JEV causes severe
central nerve system
diseases such as
p o l i o mye ...
Japanese Encephalitis Virus
          (JEV)
Japanese Encephalitis Virus
          (JEV)
Japanese Encephalitis Virus
          (JEV)



50,000 Cases
Japanese Encephalitis Virus
          (JEV)



30% fatality rate
Japanese Encephalitis Virus
          (JEV)



10,000 Cases
Prevention and
treatment of JEV disease
Prevention and
treatment of JEV disease

   Drug          No drug exist
Prevention and
 treatment of JEV disease

       Drug            No drug exist


Vaccine development   Available vaccine
Prevention and
 treatment of JEV disease

       Drug                No drug exist


Vaccine development       Available v...
Molecular biology of Japanese
     Encephalitis Virus
The NS2B
• 130 aa
• activating domain
    central hydrophilic region
    (Falgout et al, 1993)
• 3 membrane spanning parts
The NS2B
• 130 aa
• activating domain
    central hydrophilic region
    (Falgout et al, 1993)
• 3 membrane spanning parts...
The NS2B
  • 130 aa
  • activating domain
      central hydrophilic region
      (Falgout et al, 1993)
  • 3 membrane span...
The NS2B
  • 130 aa                         Hypothetical model
  • activating domain              NS2B-NS3 complex
      c...
The NS3




Theoretical model from PDB
            2I84
The NS3

                             Protease




Theoretical model from PDB
            2I84
The NS3

                             Protease

                             NTPase



                             RNA He...
The NS3




                             •Chymotrypsin-like fold
                             2-β barrel domains
         ...
The NS3 protease
The NS3 protease


       •NS3 serine protease
       domain 20 kDa
       •catalytic residues His51,
       Asp75, Ser135
Background




      • Lin. C W et al,2007
Background
• Ser  to Ile60 were essential region required for NS3
        46
  protease activity.
• Ala substition of Trp
...
Objective
Objective
•   to perform cloning of the NS2B-NS3 portion of the JEV
    polyprotein, express in E.coli and biochemically p...
Method & Result
pLS with NS2B-NS3 JEV
Method & Result
pLS with NS2B-NS3 JEV

NS2B(H)      NS3p
Method & Result
pLS with NS2B-NS3 JEV

NS2B(H)      NS3p


      SOE-PCR
Method & Result
pLS with NS2B-NS3 JEV

NS2B(H)      NS3p


      SOE-PCR

    NS2B(H)-NS3p
NS2B(H)

                                        NS2B(H)
                    Control




                                 ...
Control
               NS2B(H)-NS3p




                                                                                  ...
Method & Result
Method & Result
NS2B(H)-NS3p JEV             pTrcHis A




         Ligation & Transformation


 Site Screening & Digest w...
23.13 kb




2.03 kb
2.32 kb
           4.56 kb
           6.56 kb
           9.42 kb
                     pTrc plasmid
  ...
Digest with Restriction Enzyme




                                                             clone 1 undigested
       ...
Sequencing of candidate
clone.
Deduced amino acid sequences of
Japanese encephalitis virus

             NS2B(H)
                              NS2B(H) C-...
Expression of Japanese encephalitis virus
NS2B(H)/NS3 protease at different time.

                                       ...
SDS-PAGE Analysis of Japanese encephalitis
virus NS2B(H)/NS3 protease at different time.
             marker


           ...
Western blot analysis of Japanese encephalitis virus
NS2B(H)/NS3 protease at different time.

              marker



    ...
Expression of Japanese encephalitis virus
  NS2B(H)/NS3 protease expressed at
  different temperatures.

              1% ...
SDS-PAGE analysis of Japanese encephalitis virus
NS2B(H)/NS3 protease expressed at different
temperatures.


             ...
Western blot analysis of Japanese encephalitis
virus NS2B(H)/NS3 protease expressed at
different temperatures.




       ...
Expression of Japanese encephalitis virus
  NS2B(H)/NS3 protease expressed at different
  IPTG concentrations
            ...
SDS-PAGE analysis of Japanese encephalitis virus
NS2B(H)/NS3 protease expressed at different IPTG
concentrations.




    ...
Western blot analysis of Japaneseencephalitis
  virus NS2B(H)/NS3 protease expressed at
  different IPTG concentrations

 ...
Expression of Japanese encephalitis virus
NS2B(H)/NS3 protease at small scale
expression.
Expression of Japanese encephalitis virus
 NS2B(H)/NS3 protease at small scale
 expression.


              1%            ...
SDS-PAGE analysis of Japanese encephalitis
virus NS2B(H)/NS3 protease at small scale
expression.
           marker
       ...
Western blot analysis of Japanese encephalitis
virus NS2B(H)/NS3 protease at small scale
expression.


                   ...
SDS-PAGE analysis of Japanese encephalitis
  virus NS2B(H)/NS3 protease at large scale
  expression.
          marker




...
Western blot analysis of Japanese encephalitis
virus NS2B(H)/NS3 protease at large scale
expression.
 marker



          ...
Purification of the NS2B(H)/NS3p protein
harboring the N-terminal polyhistidine tag

  The NS2B(H)/NS3 protease was purify ...
Method
Soluble fraction in buffer H




            Wash with Buffer H (30 mM imidazole)



            Elute with Buffer ...
SDS-PAGE analysis of Japanese encephalitis virus
NS2B(H)/NS3 protease from Hi-trap purification.




                      ...
Western-blot analysis of Japanese encephalitis virus
NS2B(H)/NS3 protease from
Hi-trap column purification




            ...
Characterize the activity of NS2B(H)/
NS3p JEV with Ac-RRRR-pNA
 The substrate Ac-RRRR-pNA is based on the para-
 nitroani...
Result
                                                      Trypsin




                                              NS2...
Problem


• The protein has by products from the
  purification step.

• No activity
• The fusion protein was different fro...
The International Journal of Biochemistry & Cell
          Biology 39 (2007) 606–614
Method
Soluble fraction in buffer A
        (50mM Hepes pH 7.0, 500mM NaCl)




              Wash with Buffer A (30 mM im...
Method

The eluted protein

                     (superdex 75 HR 10/300)
peak1
peak2
101 kD
                                             210 kD
                                             125 kD




6.9 kD
...
29 kD




         21 kD
                                             101 kD
                                             ...
29 kD




         21 kD
                                             101 kD
                                             ...
29 kD



         21 kD
                                             101 kD
                                             2...
29 kD

         21 kD
                                             101 kD
                                             210...
The substrate
The substrate
Protein assay method
 7-Amino-4-Methyl Coumarin (AMC) standard curve


• A serial dilution of AMC, from 3 μM -
   50 μM, w...
result

                                            Data 1
                          100000
                              ...
Protein assay method
Enzyme Activity assay

NS2B(H)-NS3p JEV protein 100 μl
assay buffer (200 mM Tris-HCl pH. 9.5,
   13.5...
result
                                              Data 1
                             30000
                           ...
Protein assay method
     Determination of kinetic parameters

NS2B(H)-NS3p JEV protein 100 μl
assay buffer (200 mM Tris-H...
result
                                                         Data 1
                               2000


             ...
What’s next?

• Try to improve purification and
  find the amount of active
  protein

• compare to Den NS2B(H)-NS3p
Evaluation 3
Evaluation 3
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Evaluation 3

  1. 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. 2. Japanese Encephalitis Virus (JEV) -Flaviviridae family -Mosquito-borne neurotropic flavivirus •causes severe central nerve system diseases
  3. 3. Japanese Encephalitis Virus (JEV) Culex tritaeniorhynchus. Source: fehd.gov.hk
  4. 4. Japanese Encephalitis Virus (JEV)
  5. 5. Japanese Encephalitis Virus (JEV) Source : vietnammedicalpractice.com
  6. 6. Japanese Encephalitis Virus (JEV)
  7. 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. 8. Japanese Encephalitis Virus (JEV)
  9. 9. Japanese Encephalitis Virus (JEV)
  10. 10. Japanese Encephalitis Virus (JEV) 50,000 Cases
  11. 11. Japanese Encephalitis Virus (JEV) 30% fatality rate
  12. 12. Japanese Encephalitis Virus (JEV) 10,000 Cases
  13. 13. Prevention and treatment of JEV disease
  14. 14. Prevention and treatment of JEV disease Drug No drug exist
  15. 15. Prevention and treatment of JEV disease Drug No drug exist Vaccine development Available vaccine
  16. 16. Prevention and treatment of JEV disease Drug No drug exist Vaccine development Available vaccine Elimination of mosquitoes Mosquitoes control breeding places
  17. 17. Molecular biology of Japanese Encephalitis Virus
  18. 18. The NS2B • 130 aa • activating domain central hydrophilic region (Falgout et al, 1993) • 3 membrane spanning parts
  19. 19. The NS2B • 130 aa • activating domain central hydrophilic region (Falgout et al, 1993) • 3 membrane spanning parts hydrophobicity plot
  20. 20. The NS2B • 130 aa • activating domain central hydrophilic region (Falgout et al, 1993) • 3 membrane spanning parts hydrophobicity plot 51 DMWLERAADISWEMDAAITGSSRRLDVKLDDDGDFHLIDDPGVP 95
  21. 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. 22. The NS3 Theoretical model from PDB 2I84
  23. 23. The NS3 Protease Theoretical model from PDB 2I84
  24. 24. The NS3 Protease NTPase RNA Helicase Theoretical model from PDB 2I84
  25. 25. The NS3 •Chymotrypsin-like fold 2-β barrel domains •Inactive alone Theoretical model from PDB •Enzyme’s pocket is small 2I84
  26. 26. The NS3 protease
  27. 27. The NS3 protease •NS3 serine protease domain 20 kDa •catalytic residues His51, Asp75, Ser135
  28. 28. Background • Lin. C W et al,2007
  29. 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. 30. Objective
  31. 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. 32. Method & Result pLS with NS2B-NS3 JEV
  33. 33. Method & Result pLS with NS2B-NS3 JEV NS2B(H) NS3p
  34. 34. Method & Result pLS with NS2B-NS3 JEV NS2B(H) NS3p SOE-PCR
  35. 35. Method & Result pLS with NS2B-NS3 JEV NS2B(H) NS3p SOE-PCR NS2B(H)-NS3p
  36. 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. 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. 38. Method & Result
  39. 39. Method & Result NS2B(H)-NS3p JEV pTrcHis A Ligation & Transformation Site Screening & Digest with Restriction Enzyme
  40. 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. 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. 42. Sequencing of candidate clone.
  43. 43. Deduced amino acid sequences of Japanese encephalitis virus NS2B(H) NS2B(H) C-terminal NS3p
  44. 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. 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. 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. 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. 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. 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. 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. 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. 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. 53. Expression of Japanese encephalitis virus NS2B(H)/NS3 protease at small scale expression.
  54. 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. 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. 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. 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. 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. 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. 60. Method Soluble fraction in buffer H Wash with Buffer H (30 mM imidazole) Elute with Buffer H (100 mM imidazole)
  61. 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. 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. 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. 64. Result Trypsin NS2B(H)/NS3p JEV substrate NS2B(H)/NS3p JEV = 10 μM trypsin = 0.4 μM substrate = 500 μM
  65. 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. 66. The International Journal of Biochemistry & Cell Biology 39 (2007) 606–614
  67. 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. 68. Method The eluted protein (superdex 75 HR 10/300)
  69. 69. peak1 peak2
  70. 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. 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. 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. 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. 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. 75. The substrate
  76. 76. The substrate
  77. 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. 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. 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. 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. 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. 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. 83. What’s next? • Try to improve purification and find the amount of active protein • compare to Den NS2B(H)-NS3p

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