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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
Japanese Encephalitis Virus
             Flaviviridae family
Dengue (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

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
Prevention and treatment of
JEV disease
       Drug            No drug exist

Vaccine development   Available vaccine

 Mosquitoes control    Elimination of
                        mosquitoes
                      breeding places
Molecular biology of
Japanese Encephalitis Virus




                 www. molecular-virology.uni-hd.de
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
The NS3
                                 Protease

                                     NTPase




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


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

                                  Jan. L R et al, 1995
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
Method & Result
pLS with NS2B-NS3 JEV

NS2B(H)      NS3p

      SOE-PCR

    NS2B(H)-NS3p
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.
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.
Method & Result
NS2B(H)-NS3p JEV             pTrcHis A




         Ligation & Transformation


 Site Screening & Digest with Restriction Enzyme
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
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
Sequencing of candidate
clone.
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
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
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.
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
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.
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
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.
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.
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
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
 NS2B(H)             NS3p




           A. N. Warrens et al. 1996
NS2B(H)-NS3p JE
MW from Expasy
pTrcHis A




      Text

             invitrogen

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Seminar 2

  • 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
  • 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
  • 36. pTrcHis A Text invitrogen