with uncorrected grammar and misspelling

1. Cloning, Expression, Purification and
Enzymological Characterization of
NS2B(H)/NS3 protease of
Japanese Encephalitis Virus.
Chakard Chalayut
Advisor: Assit. Prof. Gerd Katzenmier
Laboratory of Molecular Virology
Institute of Molecular Biology & Genetics

2. Japanese Encephalitis Virus
Flaviviridae family
Dengue (Den)
West nile virus (WNV)
Yellow fever virus (YFV)
Japanese Encephalitis Virus (JEV)
ETC...

3. Japanese Encephalitis Virus
Mosquito-borne neurotropic flavivirus
causes severe central nerve system diseases
Divided into 4 Genotypes.
For unknown reason genotypes 3 is the most outbreak.
Culex tritaeniorhynchus is the important vector.
http://www.fehd.gov.hk

4. Japanese Encephalitis Virus
J E V c a u s e s s e v e re
central nerve system
50,000 cases/year
30% fatality rate
10,000 Death Cases/year
diseases such as
poliomyelitis- like acute
flaccid paralysis, aseptic
m e n i n g i t i s a n d http://www.wonder.cdc.gov
encephalitis
http://www.cdc.gov

5. Prevention and treatment of
JEV disease
Drug No drug exist
Vaccine development Available vaccine
Mosquitoes control Elimination of
mosquitoes
breeding places

6. Molecular biology of
Japanese Encephalitis Virus
www. molecular-virology.uni-hd.de

7. The NS2B
130 aa
activating domain central hydrophilic region
(Falgout et al, 1993)
3 membrane spanning parts Hypothetical model
NS2B-NS3 complex
58 VSGKATDMWLERAADISWEMDAAITGSSRRLDVKLDDDGDFHLIDDPGVP 101
Brinkworth et al, 1999

8. The NS3
Protease
NTPase
RNA Helicase
Chymotrypsin-like fold
2-β barrel domains
Inactive alone
Theoretical modelEnzyme’s pocket is small
from PDB
2I84

9. The NS3 protease
Complexation with NS2B cofactor
NS3 serine protease
domain 20 kDa
catalytic residues His51,
Asp75, Ser135
conformational change
alteration of the enzyme
pocket additional substrate
binding site

10. 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.

11. Background
NS2B JE - NS3 Den did not cleaved Den polyprotein but
NS2B Den - NS3 JE cleaved JEV polyprotein.
The C-terminal portion of Den NS2B is required
forinteraction with Den NS3 to activate protease.
Jan. L R et al, 1995

12. Background
Ser46 to Ile60 were essential region required for NS3
protease activity.
Ala substition of Trp50, Glu55, and Arg56 in NS2B shown
significantly reduced NS3 protease activity.
Lin. C W et al,2007

13. Method & Result
pLS with NS2B-NS3 JEV
NS2B(H) NS3p
SOE-PCR
NS2B(H)-NS3p

14. 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.

15. 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.

16. Method & Result
NS2B(H)-NS3p JEV pTrcHis A
Ligation & Transformation
Site Screening & Digest with Restriction Enzyme

17. 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

18. 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

19. Sequencing of candidate
clone.

20. Expression Condition
By varied time.
0 Hr
1% 2 Hr
4 Hr
6 Hr
incubate overnight
O.D.= 0.4-0.6 8 Hr
induction by IPTG

21. 101 kD
125 kD
210 kD
35.8 kD
56.2 kD
21 kD
29 kD
6.9 kD
marker
pTrc 0 hr
pTrc 2 hr
pTrc 4 hr
Result
pTrc 8 hr
pTrcNS2B(H)/NS3p 0 hr
pTrcNS2B(H)/NS3p 2 hr
pTrcNS2B(H)/NS3p 4 hr
pTrcNS2B(H)/NS3p 6 hr
pTrcNS2B(H)/NS3p 8 hr
marker
pTrcNS2B(H)/NS3p 0 hr
pTrcNS2B(H)/NS3p 2 hr
pTrcNS2B(H)/NS3p 4 hr
pTrcNS2B(H)/NS3p 6 hr
pTrcNS2B(H)/NS3p 8 hr

22. Expression Condition
By varied temperature.
1% 18 ˚C
25 ˚C
37 ˚C
incubate overnight
O.D.= 0.4-0.6
induction by IPTG
Incubate 8 Hrs.

23. 101 kD
125 kD
210 kD
21 kD
29 kD
6.9 kD
35.8 kD
56.2 kD
marker
pTrc 0 hr 37 ˚C
pTrcNS2B(H)/NS3p 25 ˚C
Result
pTrc 25 ˚C
pTrcNS2B(H)/NS3p 18 ˚C
pTrc 18 ˚C
pTrcNS2B(H)/NS3p 37 ˚C
pTrc 37 ˚C
pTrcNS2B(H)/NS3p 30 ˚C
pTrc 30 ˚C
marker
pTrc 0 hr 37 ˚C
pTrcNS2B(H)/NS3p 18 ˚C
pTrc 18 ˚C
pTrcNS2B(H)/NS3p 25 ˚C
pTrc 25 ˚C
pTrcNS2B(H)/NS3p 37 ˚C
pTrc 37 ˚C
pTrcNS2B(H)/NS3p 30 ˚C
pTrc 30 ˚C

24. Expression Condition
By varied IPTG concentration
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.

25. 6.9 kD
125 kD
101 kD
210 kD
21 kD
29 kD
35.8 kD
56.2 kD
marker
pTrc 0.1 mM
Result
pTrcNS2B(H)/NS3p 0.1 mM
pTrcNS2B(H)/NS3p 0.2 mM
pTrcNS2B(H)/NS3p 0.3 mM
pTrcNS2B(H)/NS3p 0.4 mM
pTrcNS2B(H)/NS3p 0.5 mM
pTrcNS2B(H)/NS3p 0.6 mM
pTrcNS2B(H)/NS3p 0.7 mM
pTrcNS2B(H)/NS3p 0.8 mM
marker
pTrc 0.1 mM
pTrcNS2B(H)/NS3p 0.1 mM
pTrcNS2B(H)/NS3p 0.2 mM
pTrcNS2B(H)/NS3p 0.3 mM
pTrcNS2B(H)/NS3p 0.4 mM
pTrcNS2B(H)/NS3p 0.5 mM
pTrcNS2B(H)/NS3p 0.6 mM
pTrcNS2B(H)/NS3p 0.7 mM
pTrcNS2B(H)/NS3p 0.8 mM

26. Conclusion.
The NS2B(H)/NS3 DNA was successfully constructed
and cloned into pTrcHis A expression vector.
The NS2B(H)/NS3 protease have the high expression
level after induction with 0.1mM IPTG 8 hour in 37 ˚C.

27. Expression condition on
Now...
solubilization
1% Centrifuge,
Lysis,
Find the expression condition Sonication
soluble or inclusion body fraction? and Collect
incubate overnight fraction
O.D.= 0.4-0.6
induction by o.1 mM IPTG
Incubate 8 Hrs.

28. 6.9 kD
125 kD
101 kD
210 kD
21 kD
29 kD
35.8 kD
56.2 kD
marker
Total fraction
Result
Soluble fraction
Inclusion fraction
marker
Total fraction
Soluble fraction
Inclusion fraction
marker
Total fraction
Soluble fraction
Inclusion fraction
marker
Total fraction
Soluble fraction
Inclusion fraction

29. Future work
The NS2B(H)/NS3 protease will further purify by HiTrap
Chelating column.

30. Future work
Characterize the activity on Cis and Trans Cleavage.
Cis Clevage : the self cleavage on NS2B(H) and
NS3p cleavage site.
Trans Cleavage :the protease activity on synthetic
substrate GRR-AMC.
excitation wavelength = 355 nm
emission wavelength = 460 nm.

31. Additional work
Construct recombinant NS2B(H) and NS3 protease of
Dengue and Japanese encephalitis virus.
JEV Den

32. Thank you for your attention.

33. SOE-PCR
NS2B(H) NS3p
A. N. Warrens et al. 1996

34. NS2B(H)-NS3p JE

35. MW from Expasy

36. pTrcHis A
Text
invitrogen