evaluation i.key

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

Japanese Encephalitis Virus

-Flaviviridae family-Mosquito-borne neurotropic flavivirus

•causes severe central nerve system diseases

Japanese Encephalitis Virus

Culex tritaeniorhynchus.

Source: fehd.gov.hkSource : vietnammedicalpractice.com

Japanese Encephalitis Virus

JEV causes severe central nerve system d i s e a s e s s u c h a s po l iomye l i t i s - l i ke acute flaccid paralysis, aseptic meningitis and encephalitis

Source:wonder.cdc.gov

Source: cdc.gov

30% fatality rate50,000 Cases10,000 Cases

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

Source : molecular-virology.uni-hd.de

The NS2BHypothetical model NS2B-NS3 complex

hydrophobicity plot

51 DMWLERAADISWEMDAAITGSSRRLDVKLDDDGDFHLIDDPGVP 95

Brinkworth et al, 1999

• 130 aa• activating domain central hydrophilic region (Falgout et al, 1993)• 3 membrane spanning parts

The NS3

•Chymotrypsin-like fold2-β barrel domains •Inactive alone•Enzyme’s pocket is small

NTPase

Protease

RNA Helicase

Theoretical model from PDB 2I84

The NS3 protease

conformational change alteration of the enzyme pocket additional substrate binding site

•NS3 serine protease domain 20 kDa•catalytic residues His51, Asp75, Ser135

Complexation with NS2B cofactor

Background• NS2B(H) JE - NS3p Den did not cleaved Den

polyprotein but NS2B(H) Den - NS3p JE cleaved JEV polyprotein.

• Jan. L R et al, 1995

• The C-terminal portion of Den NS2B is required for interaction with Den NS3 to activate protease.

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

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 subst ra tes incorporated wi th fluorogenic or chromogenic reported groups.

Method & Result pLS with NS2B-NS3 JEV

NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p

NS2B(H) NS3p NS3NS2B

NS3pNS2B(H)

1500

1000900800700

600500

400

300

200

-C

ontrol

NS2B

(H)

NS2B

(H)

1500

1000900800700600

500

400

300

200

-C

ontrol

NS3 protease

NS3 protease

NS3 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

1000900800700600

500400

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.

NS2B

(H)-N

S3p

-C

ontrol

1500

1000900800700600

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.

-C

ontrol

NS2B

(H)-N

S3p

Method & Result pTrcHis Den with NS2B-NS3 Den

NS2B(H) NS3p

SOE-PCR

NS2B(H)-NS3p Den

NS2B

(H)

NS2B

(H)

-C

ontrol

1500

1000900800700600500

400

300

200

100

Figure5 : The PCR product NS2B(H) Den amplified from pTrc NS2B-NS3 Den (Lane 2 and 3). The size of NS2B(H) was 187 bp.

-C

ontrol

NS3 protease

NS3 protease

1500

1000900800700600500

400

300

200

100

Figure 6 : The PCR product NS3protease Den amplified from pTrc NS2B-NS3 Den (Lane 3 and 4 ). The size of NS3 protease was 594 bp.

NS2B

(H)-N

S3p

NS2B

(H)-N

S3p

NS2B

(H)-N

S3p

NS2B

(H)-N

S3p

NS2B

(H)-N

S3p

-C

ontrol

1500

1000900800700600500

400

300

200

Figure 7 : The SOE-PCR product NS2B(H)-NS3protease Den (Lane 2 and 6). The size of NS2B(H)-NS3 protease was 765 bp.

-C

ontrol

NS2B

(H)-N

S3p

NS2B

(H)-N

S3p

1500

1000900800700600500

400

300

200

Figure 8 :The NS2B(H)-NS3protease Den (Lane 3 and 4 ) after digested with BamHI and KpnI. The size of NS2B(H)-NS3 protease was 765 bp.

Method & ResultpTrcHis A

Digest with BamHI Digest with KpnI

Digest with BamHIDigest with KpnI

Gel Electrophoresis & Clean with Gel Extraction Kits

23.13 kb9.42 kb6.56 kb4.56 kb

2.32 kb2.03 kb

Figure 9 : The pTrcHis A in lane 2 without digestion

23.13 kb9.42 kb6.56 kb4.56 kb

2.32 kb2.03 kb

pTrcHis A

Digested pTrcH

is A

with Bam

HI

Digested pTrcH

is A

with K

pnI

Figure 10 : The pTrcHis A in lane digested with BamHI and KpnI in lane 3 and 4.In line 1 is pTrcHis A without digestion.

Method & Result

NS2B(H)-NS3p Den

NS2B(H)-NS3p JEVpTrcHis A

Ligation & Transformation

Site Screening & Digest with Restriction Enzyme

Dengue C

lone 1-5

Dengue C

lone 6-10

Dengue C

lone 11-15

Dengue C

lone 16-20

Dengue C

lone 21-25

JEV C

lone 1

pTrcHis A

23.13 kb

9.42 kb6.56 kb4.56 kb

2.32 kb2.03 kb

Figure 11 : The pTrcHis NS2B(H)-NS3protease JEV (lane 8) and pTrcHis NS2B(H)-NS3protease Den (lane 3 to 7) was digested with BamHI.

23.13 kb

9.42 kb6.56 kb4.56 kb

2.32 kb2.03 kb

700 bpN

S2B(H

)-NS3p

Dengue C

lone 1

Dengue C

lone 2

Dengue C

lone 3

Dengue C

lone 4

Dengue C

lone 5

Dengue C

lone 6

Dengue C

lone 7

Dengue C

lone 8

Dengue C

lone 9

JEV C

lone 1

Figure 12 : The pTrcHis NS2B(H)-NS3protease JEV (lane 12) and pTrcHis NS2B(H)-NS3protease Den clone 1-9 (lane 3 to 11) was digested with BamHI and KpnI.

Conclusion

• The NS2B(H)-NS3p JEV and NS2B(H)-NS3p Den was successfully PCR and ligated into pTrcHis A plasmid.

• The digestion of the recombinant vector shown the expect band of pTrcHis with the insert, then it need to purify and sequence the plasmid to make sure,that is the correct plasmid.

Future Work

• Check another clone to get more positive clone.

• Construct NS2B(H)Den-NS3p JE and NS2B(H) JE-NS3p Den.

• Retransform into E.coli C41.

• Purification of Protein.

• Enzyme assay.

Thank you for your attention.