seminar 2
DESCRIPTION
with uncorrected grammar and misspellingTRANSCRIPT
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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
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Japanese Encephalitis Virus
Flaviviridae familyDengue (Den)
West nile virus (WNV)
Yellow fever virus (YFV)
Japanese Encephalitis Virus (JEV)
ETC...
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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
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Japanese Encephalitis Virus
JEV causes severe central nerve system d i s e a s e s s u c h a s poliomyelitis- like acute flaccid paralysis, aseptic m e n i n g i t i s a n d encephalitis
http://www.wonder.cdc.gov
http://www.cdc.gov
50,000 cases/year30% fatality rate10,000 Death Cases/year
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Prevention and treatment of JEV disease
Drug No drug exist
Vaccine development
Mosquitoes control Elimination of mosquitoes
breeding places
Available vaccine
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Molecular biology of Japanese Encephalitis Virus
www. molecular-virology.uni-hd.de
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The NS2B130 aa
activating domain central hydrophilic region (Falgout et al, 1993)
3 membrane spanning parts Hypothetical model NS2B-NS3 complex
Brinkworth et al, 199958 VSGKATDMWLERAADISWEMDAAITGSSRRLDVKLDDDGDFHLIDDPGVP 101
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The NS3
Chymotrypsin-like fold2-β barrel domains
Inactive aloneEnzyme’s pocket is small
NTPase
Protease
RNA Helicase
Theoretical model from PDB 2I84
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The NS3 protease
conformational changealteration of the enzyme pocket additional substrate binding site
NS3 serine protease domain 20 kDacatalytic residues His51, Asp75, Ser135
Complexation with NS2B cofactor
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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.
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Background
NS2B JE - NS3 Den did not cleaved Den polyprotein but NS2B Den - NS3 JE cleaved JEV polyprotein.
Jan. L R et al, 1995
The C-terminal portion of Den NS2B is required forinteraction with Den NS3 to activate protease.
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BackgroundSer46 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
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Method & Result pLS with NS2B-NS3 JEV
NS2B(H) NS3p
SOE-PCR
NS2B(H)-NS3p
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1500
1000900800700
600
500
400
300
200
Control
NS
2B(H
)
NS
2B(H
)
1500
1000900800
700
600
500
400
300
200
Control
NS
3 protease
NS
3 protease
NS
3 protease
Figure 2 : The PCR product NS3protease JEV amplified from NS2B-NS3 JEV (Lane 3 to 5 ). The size of NS3 protease was 594 bp.
Figure 1 : The PCR product NS2B(H) JEV amplified from NS2B-NS3 JEV (Lane 3 and 4). The size of NS2B(H) was 187 bp.
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1500
1000900800
700
600
500
400
300
Figure 3 : The SOE-PCR product NS2B(H)-NS3protease JEV (Lane 2 to 5 ). The size of NS2B(H)-NS3 protease was 765 bp.
Control
1500
1000900800700
600
500
400
300
200
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.
Control
NS
2B(H
)-NS
3p
NS2B(H)-NS3p
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Method & Result
NS2B(H)-NS3p JEV pTrcHis A
Ligation & Transformation
Site Screening & Digest with Restriction Enzyme
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Size Screening
23.13 kb
9.42 kb 6.56 kb 4.56 kb
2.32 kb 2.03 kb
pTrc
pla
smid
Con
trol
Clo
ne 1
Clo
ne 3
Clo
ne 4
Clo
ne 5
Clo
ne 6
Clo
ne 7
Clo
ne 8
Clo
ne 9
Clo
ne 1
0C
lone
11
Clo
ne 1
2C
lone
13
Clo
ne 1
4
Clo
ne 1
5C
lone
16
Clo
ne 1
7
Clo
ne 2
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Digest with Restriction Enzyme
3.645 kb
2.323 kb 1.929 kb
1.371 kb 1.264 kb
702 bp
4.324 kb4.822 kb5.686 kb6.369 kb7.242 kb8.454 kb
clon
e 1
with
Bam
HI,K
pnI
clon
e 1
with
Bam
HI
clon
e 1
undi
gest
ed
•Lane 1 : λ/BstII marker
•Lane 2 : Clone 1 with BamHI and KpnI digerstion
•Lane 3 : Clone 1 with BamHI digestion
•Lane 4 : Clone 1 without digestion
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Sequencing of candidate clone.
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Expression ConditionBy varied time.
0 Hr
2 Hr
4 Hr
6 Hr
8 HrO.D.= 0.4-0.6 induction by IPTG
incubate overnight
1%
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Result
210 kD125 kD101 kD56.2 kD
35.8 kD
29 kD
21 kD
6.9 kD
pTrc
0 h
r
pTrc
2 h
r
pTrc
4 h
r
pTrc
8 h
r
pTrc
NS
2B(H
)/NS
3p 0
hr
pTrc
NS
2B(H
)/NS
3p 2
hr
pTrc
NS
2B(H
)/NS
3p 4
hr
pTrc
NS
2B(H
)/NS
3p 6
hr
pTrc
NS
2B(H
)/NS
3p 8
hr
pTrc
NS
2B(H
)/NS
3p 0
hr
pTrc
NS
2B(H
)/NS
3p 2
hr
pTrc
NS
2B(H
)/NS
3p 4
hr
pTrc
NS
2B(H
)/NS
3p 6
hr
pTrc
NS
2B(H
)/NS
3p 8
hr
mar
ker
mar
ker
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Expression ConditionBy varied temperature.
O.D.= 0.4-0.6 induction by IPTGIncubate 8 Hrs.
incubate overnight
1% 18 ˚C
37 ˚C
25 ˚C
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Result
210 kD125 kD101 kD56.2 kD
35.8 kD
29 kD
21 kD
6.9 kD
pTrc
0 h
r 37
˚C
pTrc
25
˚C
pTrc
18
˚C
pTrc
NS
2B(H
)/NS
3p 1
8 ˚C
pTrc
NS
2B(H
)/NS
3p 3
7 ˚C
pTrc
NS
2B(H
)/NS
3p 2
5 ˚C
pTrc
NS
2B(H
)/NS
3p 3
0 ˚C
pTrc
37
˚C
pTrc
30
˚C
mar
ker
pTrc
0 h
r 37
˚C
pTrc
25
˚C
pTrc
18
˚C
pTrc
NS
2B(H
)/NS
3p 1
8 ˚C
pTrc
NS
2B(H
)/NS
3p 3
7 ˚C
pTrc
NS
2B(H
)/NS
3p 2
5 ˚C
pTrc
NS
2B(H
)/NS
3p 3
0 ˚C
pTrc
37
˚C
pTrc
30
˚C
mar
ker
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Expression ConditionBy varied IPTG concentration
O.D.= 0.4-0.6 induction by IPTGIncubate 8 Hrs.
incubate overnight
1%
0.1 mM0.2 mM0.3 mM0.4 mM
0.8 mM0.7 mM0.6 mM
0.5 mM
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Result
210 kD
125 kD101 kD
56.2 kD
35.8 kD
29 kD
21 kD
6.9 kD
mar
ker
pTrc
0.1
mM
pTrc
NS
2B(H
)/NS
3p 0
.1 m
M
pTrc
NS
2B(H
)/NS
3p 0
.5 m
M
pTrc
NS
2B(H
)/NS
3p 0
.6 m
M
pTrc
NS
2B(H
)/NS
3p 0
.7 m
M
pTrc
NS
2B(H
)/NS
3p 0
.8 m
M
pTrc
NS
2B(H
)/NS
3p 0
.3 m
M
pTrc
NS
2B(H
)/NS
3p 0
.4 m
M
pTrc
NS
2B(H
)/NS
3p 0
.2 m
M
mar
ker
pTrc
0.1
mM
pTrc
NS
2B(H
)/NS
3p 0
.1 m
M
pTrc
NS
2B(H
)/NS
3p 0
.5 m
M
pTrc
NS
2B(H
)/NS
3p 0
.6 m
M
pTrc
NS
2B(H
)/NS
3p 0
.7 m
M
pTrc
NS
2B(H
)/NS
3p 0
.8 m
M
pTrc
NS
2B(H
)/NS
3p 0
.3 m
M
pTrc
NS
2B(H
)/NS
3p 0
.4 m
M
pTrc
NS
2B(H
)/NS
3p 0
.2 m
M
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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.
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Now...
O.D.= 0.4-0.6 induction by o.1 mM IPTG
Incubate 8 Hrs.
incubate overnight
1% Centrifuge, Lysis,
Sonication and Collect
fraction
Expression condition on solubilization
Find the expression condition
soluble or inclusion body fraction?
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210 kD
125 kD101 kD
56.2 kD
35.8 kD
29 kD
21 kD
6.9 kD
mar
ker
mar
ker
Tota
l fra
ctio
n
Sol
uble
frac
tion
Sol
uble
frac
tion
Tota
l fra
ctio
n
Incl
usio
n fra
ctio
n
Incl
usio
n fra
ctio
n
mar
ker
mar
ker
Tota
l fra
ctio
n
Sol
uble
frac
tion
Sol
uble
frac
tion
Tota
l fra
ctio
n
Incl
usio
n fra
ctio
n
Incl
usio
n fra
ctio
n
Result
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Future work The NS2B(H)/NS3 protease will further purify by HiTrap Chelating column.
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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.
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Additional work
Construct recombinant NS2B(H) and NS3 protease of Dengue and Japanese encephalitis virus.
JEV Den
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Thank you for your attention.
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SOE-PCR
A. N. Warrens et al. 1996
NS2B(H) NS3p
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NS2B(H)-NS3p JE
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MW from Expasy
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pTrcHis A
invitrogen
Text