my thesis evaluation3

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

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

55. 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.

56. 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

57. 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)

58. 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)

59. 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)

60. Purification of the NS2B(H)/NS3p protein
harboring the N-terminal polyhistidine tag
The NS2B(H)/NS3 protease was purify by HiTrap
Chelating column.

61. Method
Soluble fraction in buffer H
Wash with Buffer H (30 mM imidazole)
Elute with Buffer H (100 mM imidazole)

62. 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

63. 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

64. 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

65. Result
Trypsin
NS2B(H)/NS3p JEV
substrate
NS2B(H)/NS3p JEV = 10 μM trypsin = 0.4 μM substrate = 500 μM

66. Problem
• The protein has by products from the
purification step.
• No activity
• The fusion protein was different from
the previous work of JEV.

67. The International Journal of Biochemistry & Cell
Biology 39 (2007) 606–614

68. 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)

69. Method
The eluted protein
(superdex 75 HR 10/300)

70. peak1
peak2

71. 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

73. 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

74. 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

75. 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

76. 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

77. The substrate

78. The substrate

79. 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

80. 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]

81. 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.

82. 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

83. 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.

84. 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

85. What’s next?
• Try to improve purification and
find the amount of active
protein
• compare to Den NS2B(H)-NS3p