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

Japanese Encephalitis Virus

Flaviviridae familyDengue (Den)

West nile virus (WNV)

Yellow fever virus (YFV)

Japanese Encephalitis Virus (JEV)

ETC...

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

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

Prevention and treatment of JEV disease

Drug No drug exist

Vaccine development

Mosquitoes control Elimination of mosquitoes

breeding places

Available vaccine

Molecular biology of Japanese Encephalitis Virus

www. molecular-virology.uni-hd.de

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

The NS3

Chymotrypsin-like fold2-β barrel domains

Inactive aloneEnzyme’s pocket is small

NTPase

Protease

RNA Helicase

Theoretical model from PDB 2I84

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

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.

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.

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

Method & Result pLS with NS2B-NS3 JEV

NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

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.

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

Method & Result

NS2B(H)-NS3p JEV pTrcHis A

Ligation & Transformation

Site Screening & Digest with Restriction Enzyme

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.

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%

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

Expression ConditionBy varied temperature.

O.D.= 0.4-0.6 induction by IPTGIncubate 8 Hrs.

incubate overnight

1% 18 ˚C

37 ˚C

25 ˚C

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

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

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

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.

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?

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

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

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.

Additional work

Construct recombinant NS2B(H) and NS3 protease of Dengue and Japanese encephalitis virus.

JEV Den

Thank you for your attention.

SOE-PCR

A. N. Warrens et al. 1996

NS2B(H) NS3p

NS2B(H)-NS3p JE

MW from Expasy

pTrcHis A

invitrogen

Text