evaluation 2
TRANSCRIPT
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
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
Sequencing of candidate clone.
Deduced amino acid sequences of Japanese encephalitis virus
NS2B(H) C-terminalNS2B(H)
NS3p
Expression of Japanese encephalitis virus NS2B(H)/NS3 protease at different time.
0 Hr
2 Hr
4 Hr
6 Hr
8 HrO.D.= 0.4-0.6 induction by IPTG
incubate overnight
1%
SDS-PAGE Analysis of Japanese encephalitis virus NS2B(H)/NS3 protease at different time.
210 kD125 kD101 kD56.2 kD
35.8 kD
29 kD
21 kD
6.9 kD
mar
ker
ptrc 0- 8 Hr. NS2B(H)/NS3p 0- 8 Hr.
NS2B(H)/NS3
NS3
NS2B(H)
mar
ker
NS2B(H)/NS3p 0- 8 Hr.
35.8 kD
21 kD
29 kD
NS2B(H)/NS3 protease
NS3
NS2B(H)
Western blot analysis of Japanese encephalitis virus NS2B(H)/NS3 protease at different time.
Expression of Japanese encephalitis virus NS2B(H)/NS3 protease expressed at different temperatures.
O.D.= 0.4-0.6 induction by IPTGIncubate 8 Hrs.
incubate overnight
1% 18 ˚C
37 ˚C
25 ˚C
SDS-PAGE analysis of Japanese encephalitis virus NS2B(H)/NS3 protease expressed at different temperatures.
pTrc
37
˚C
pTrc
30
˚C
pTrc
0 h
r 37
˚C
pTrc
25
˚C
mar
ker
JEV
37
˚C
JEV
25
˚C
JEV
18
˚C
JEV
30
˚C
pTrc
18
˚C
210 kD125 kD101 kD
56.2 kD
35.8 kD
29 kD
21 kD
6.9 kD
NS2B(H)/NS3
NS3 protease
NS2B(H)
Western blot analysis of Japanese encephalitis virus NS2B(H)/NS3 protease expressed at different temperatures.
mar
ker
pTrc
0 h
r 37
˚C
pTrc
25
˚C
pTrc
18
˚C
NS
2B(H
)/NS
3p 1
8 ˚C
NS
2B(H
)/NS
3p 3
7 ˚C
NS
2B(H
)/NS
3p 2
5 ˚C
NS
2B(H
)/NS
3p 3
0 ˚C
pTrc
37
˚C
pTrc
30
˚C
101 kD
35.8 kD
6.9 kD
21 kD
NS2B(H)/NS3
NS3 protease
NS2B(H)
Expression of Japanese encephalitis virus NS2B(H)/NS3 protease expressed at different IPTG concentrations
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
SDS-PAGE analysis of Japanese encephalitis virus NS2B(H)/NS3 protease expressed at different IPTG concentrations.
210 kD125 kD
101 kD
56.2 kD
35.8 kD
29 kD
21 kD
6.9 kD
mar
ker
0.1m
M
0.8m
M
NS2B(H)/NS3
NS3 protease
NS2B(H)
Western blot analysis of Japaneseencephalitis virus NS2B(H)/NS3 protease expressed at different IPTG concentrations
mar
ker
35.8 kD
29 kDNS2B(H)/NS3 protease
0.1m
M
0.8m
M
Expression of Japanese encephalitis virus NS2B(H)/NS3 protease at small scale expression.
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 of Japanese encephalitis virus NS2B(H)/NS3 protease at small scale expression.
SDS-PAGE analysis of Japanese encephalitis virus NS2B(H)/NS3 protease at small scale expression.
210 kD125 kD
ITC S
101 kD
56.2 kD
35.8 kD
29 kD
6.9 kD
21 kD
mar
ker
Lane 1: maker Lane 2: Control pTrcHisLane 3: Total fraction (T)Lane 4: Insoluble fraction (I)Lane 5: Soluble fraction (S)
NS2B(H)/NS3 protease
NS3 protease
NS2B(H)
Western blot analysis of Japanese encephalitis virus NS2B(H)/NS3 protease at small scale expression.
IT S
NS2B(H)/NS3 protease35.8 kD
Lane 1: maker Lane 2: Control pTrcHisLane 3: Total fraction (T)Lane 4: Insoluble fraction (I)Lane 5: Soluble fraction (S)
210 kD
125 kD101 kD
56.2 kD
35.8 kD
29 kD
21 kD
6.9 kD
mar
ker
mar
ker
SDS-PAGE analysis of Japanese encephalitis virus NS2B(H)/NS3 protease at large scale expression.
T SI T SI
NS2B(H)/NS3 protease
NS3 protease
NS2B(H)
Lane 1: maker Lane 2: Total fraction (T)Lane 3: Insoluble fraction (I)Lane 4: Soluble fraction (S)
Western blot analysis of Japanese encephalitis virus NS2B(H)/NS3 protease at large scale expression.
mar
ker
mar
ker
T SI T SI
NS2B(H)/NS3 protease
NS3 protease
NS2B(H)
Lane 1: maker Lane 2: Total fraction (T)Lane 3: Insoluble fraction (I)Lane 4: Soluble fraction (S)
Purification of the NS2B(H)/NS3p protein harboring the N-terminal polyhistidine tag
The NS2B(H)/NS3 protease was purify by HiTrap Chelating column.
Method
Soluble fraction in buffer H
Wash with Buffer H (30 mM imidazole)
Elute with Buffer H (100 mM imidazole)
SDS-PAGE analysis of Japanese encephalitis virus NS2B(H)/NS3 protease from Hi-trap purification.
21 kD
NS2B(H)/NS3 protease
NS3 protease
6.9 kD
210 kD125 kD
56.2 kD
101 kD
35.8 kD
29 kD
mar
ker
Flow
trou
gh
indu
ctio
n
undu
ctio
n
Was
hing
frac
tion
Elu
te a
nd c
once
ntra
te
fract
ion
Sol
uble
frac
tion
Western-blot analysis of Japanese encephalitis virus NS2B(H)/NS3 protease from Hi-trap column purification
NS2B(H)
NS2B(H)/NS3
NS3 protease
21 kD
6.9 kD
35.8 kD
29 kD
mar
ker
Flow
trou
gh
indu
ctio
n
undu
ctio
n
Was
hing
frac
tion
Elu
te a
nd c
once
ntra
te
fract
ion
Sol
uble
frac
tion
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
Result
Trypsin
NS2B(H)/NS3p JEV
substrate
NS2B(H)/NS3p JEV = 10 μM trypsin = 0.4 μM substrate = 500 μM
Additional work
Construct recombinant NS2B(H) and NS3 protease of Dengue and Japanese encephalitis virus.
JEV Den
Sequencing of candidate recombinant Den-Jev clone .
The entire DNA sequences of JEV NS2B(H)-Den NS3p and vice-versa were analyzed by automated DNA sequencing in both forward and reverse direction using the sequencing primers.
An alignment was showed the 100% identity of JEV NS2B(H)- Den NS3p to the nucleotide sequence of JEV NS2B(H)- Den NS3p
An alignment of Japanese Encephalitis virus NS2B(H) - Dengue NS3 protease nucleotide sequence from the sequencing result and the JEV sequence.
Expression of JEV NS2B(H)- Den NS3p at different time.
0 Hr
2 Hr
4 Hr
6 Hr
8 HrO.D.= 0.4-0.6 induction by IPTG
incubate overnight
1%
SDS-PAGE Analysis of JEV NS2B(H)- Den NS3p at different time.
21 kD
6.9 kD
210 kD125 kD
56.2 kD
101 kD
35.8 kD
29 kD
mar
ker
ptrc 0- 8 Hr. NS2B(H)/NS3p 0- 8 Hr.
Western blot analysis of JEV NS2B(H)- Den NS3p at different time.
But no signals of expected protein
Plasmid digestion to check insert.
23.13 kb
9.42 kb 6.56 kb 4.56 kb
2.32 kb 2.03 kb
pTrc
pla
smid
JEV
NS
2B(H
)- D
en N
S3p
JEV
NS
2B(H
)- D
en N
S3p
sto
ck
linearizecheck insert
Conclusion
• The NS2B(H)-NS3p JEV was successfully PCR and ligated into pTrcHis A plasmid. The sequencing show 100 % identity to expected plasmid.
• The NS2B(H)-NS3p JEV was expressed in soluble form and have purified with Hi-trap columm
• The NS2B(H) Den -NS3p JEV was successfully PCR and ligated into pTrcHis A plasmid. The sequencing show 100 % identity to expected plasmid.
Problem
NS2B(H)/NS3p JEV has no activity when compared to tripsin enzyme even the purification step was done in low temperature.
The protein will be refold and characterize again
JEV NS2B(H)- Den NS3p has no expression but it had the insert.
retransform or construct a new JEV NS2B(H)- Den NS3p.
Future work
Purification of the recombinant protein
Protease activity assays, the comparison of protease activity to hybrids protein and dengue NS2B(H)/NS3p
NS2B(H) C-terminalNS2B(H)
NS3p
RR =RK = 20.309 kDaRQ = 28.884 kDa
7.029 kDa
Blast conferm