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Seminar 2
 

Seminar 2

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    Seminar 2 Seminar 2 Presentation Transcript

    • 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