Evaluation 2

Slide 1: 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

Slide 2: 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

Slide 3: Sequencing of candidate clone.

Slide 4: Deduced amino acid sequences of Japanese encephalitis virus NS2B(H) NS2B(H) C-terminal NS3p

Slide 5: Expression of Japanese encephalitis virus NS2B(H)/NS3 protease at different time. 0 Hr 1% 2 Hr 4 Hr 6 Hr incubate overnight O.D.= 0.4-0.6 8 Hr induction by IPTG

Slide 6: SDS-PAGE Analysis of Japanese encephalitis virus NS2B(H)/NS3 protease at different time. marker ptrc 0- 8 Hr. NS2B(H)/NS3p 0- 8 Hr. 210 kD 125 kD 101 kD 56.2 kD 35.8 kD NS2B(H)/NS3 29 kD NS3 21 kD 6.9 kD NS2B(H)

Slide 7: Western blot analysis of Japanese encephalitis virus NS2B(H)/NS3 protease at different time. marker NS2B(H)/NS3p 0- 8 Hr. NS2B(H)/NS3 protease 35.8 kD 29 kD 21 kD NS3 NS2B(H)

Slide 8: Expression of Japanese encephalitis virus NS2B(H)/NS3 protease expressed at different temperatures. 1% 18 ˚C 25 ˚C 37 ˚C incubate overnight O.D.= 0.4-0.6 induction by IPTG Incubate 8 Hrs.

Slide 9: SDS-PAGE analysis of Japanese encephalitis virus NS2B(H)/NS3 protease expressed at different temperatures. pTrc 0 hr 37 ˚C JEV 37 ˚C JEV 18 ˚C JEV 30 ˚C pTrc 18 ˚C pTrc 25 ˚C pTrc 37 ˚C pTrc 30 ˚C JEV 25 ˚C marker 210 kD 125 kD 101 kD 56.2 kD 35.8 kD NS2B(H)/NS3 29 kD 21 kD NS3 protease 6.9 kD NS2B(H)

Slide 10: Western blot analysis of Japanese encephalitis virus NS2B(H)/NS3 protease expressed at different temperatures. NS2B(H)/NS3p 37 ˚C NS2B(H)/NS3p 18 ˚C NS2B(H)/NS3p 30 ˚C NS2B(H)/NS3p 25 ˚C pTrc 0 hr 37 ˚C pTrc 18 ˚C pTrc 25 ˚C pTrc 37 ˚C pTrc 30 ˚C marker 101 kD 35.8 kD NS2B(H)/NS3 NS3 protease 21 kD NS2B(H) 6.9 kD

Slide 11: Expression of Japanese encephalitis virus NS2B(H)/NS3 protease expressed at different IPTG concentrations 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.

Slide 12: SDS-PAGE analysis of Japanese encephalitis virus NS2B(H)/NS3 protease expressed at different IPTG concentrations. 0.1mM 0.8mM marker 210 kD 125 kD 101 kD 56.2 kD 35.8 kD NS2B(H)/NS3 29 kD NS3 protease 21 kD 6.9 kD NS2B(H)

Slide 13: Western blot analysis of Japaneseencephalitis virus NS2B(H)/NS3 protease expressed at different IPTG concentrations 0.1mM 0.8mM marker 35.8 kD NS2B(H)/NS3 protease 29 kD

Slide 15: Expression of Japanese encephalitis virus NS2B(H)/NS3 protease at small scale expression.

Slide 16: Expression of Japanese encephalitis virus NS2B(H)/NS3 protease at small scale expression. 1% Centrifuge, Lysis, Sonication and Collect incubate overnight fraction O.D.= 0.4-0.6 induction by o.1 mM IPTG Incubate 8 Hrs.

Slide 17: SDS-PAGE analysis of Japanese encephalitis virus NS2B(H)/NS3 protease at small scale expression. marker C T I S 210 kD 125 kD 101 kD Lane 1: maker 56.2 kD NS2B(H)/NS3 protease Lane 2: Control pTrcHis Lane 3: Total fraction (T) 35.8 kD Lane 4: Insoluble fraction (I) Lane 5: Soluble fraction (S) 29 kD NS3 protease 21 kD NS2B(H) 6.9 kD

Slide 18: Western blot analysis of Japanese encephalitis virus NS2B(H)/NS3 protease at small scale expression. T I S 35.8 kD NS2B(H)/NS3 protease Lane 1: maker Lane 2: Control pTrcHis Lane 3: Total fraction (T) Lane 4: Insoluble fraction (I) Lane 5: Soluble fraction (S)

Slide 19: SDS-PAGE analysis of Japanese encephalitis virus NS2B(H)/NS3 protease at large scale expression. marker marker T I S TI S 210 kD 101 kD 125 kD 56.2 kD Lane 1: maker Lane 2: Total fraction (T) 35.8 kD NS2B(H)/NS3 protease Lane 3: Insoluble fraction (I) 29 kD Lane 4: Soluble fraction (S) NS3 protease 21 kD 6.9 kD NS2B(H)

Slide 20: Western blot analysis of Japanese encephalitis virus NS2B(H)/NS3 protease at large scale expression. marker marker TI S T I S Lane 1: maker NS2B(H)/NS3 protease Lane 2: Total fraction (T) Lane 3: Insoluble fraction (I) Lane 4: Soluble fraction (S) NS3 protease NS2B(H)

Slide 21: Purification of the NS2B(H)/NS3p protein harboring the N-terminal polyhistidine tag The NS2B(H)/NS3 protease was purify by HiTrap Chelating column.

Slide 22: Method Soluble fraction in buffer H Wash with Buffer H (30 mM imidazole) Elute with Buffer H (100 mM imidazole)

Slide 23: SDS-PAGE analysis of Japanese encephalitis virus NS2B(H)/NS3 protease from Hi-trap purification. Elute and concentrate Washing fraction Soluble fraction fraction Flowtrough unduction induction marker 210 kD 125 kD 101 kD 56.2 kD NS2B(H)/NS3 protease 35.8 kD 29 kD NS3 protease 21 kD 6.9 kD

Slide 24: Western-blot analysis of Japanese encephalitis virus NS2B(H)/NS3 protease from Hi-trap column purification Elute and concentrate Washing fraction Soluble fraction fraction unduction Flowtrough induction marker NS2B(H)/NS3 35.8 kD 29 kD NS3 protease 21 kD NS2B(H) 6.9 kD

Slide 25: 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

Slide 26: Result Trypsin NS2B(H)/NS3p JEV substrate NS2B(H)/NS3p JEV = 10 μM trypsin = 0.4 μM substrate = 500 μM

Slide 27: Additional work Construct recombinant NS2B(H) and NS3 protease of Dengue and Japanese encephalitis virus. JEV Den

Slide 28: 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

Slide 29: An alignment of Japanese Encephalitis virus NS2B(H) - Dengue NS3 protease nucleotide sequence from the sequencing result and the JEV sequence.

Slide 30: Expression of JEV NS2B(H)- Den NS3p at different time. 0 Hr 1% 2 Hr 4 Hr 6 Hr incubate overnight O.D.= 0.4-0.6 8 Hr induction by IPTG

Slide 31: SDS-PAGE Analysis of JEV NS2B(H)- Den NS3p at different time. marker ptrc 0- 8 Hr. NS2B(H)/NS3p 0- 8 Hr. 210 kD 125 kD 101 kD 56.2 kD 35.8 kD 29 kD 21 kD 6.9 kD

Slide 32: Western blot analysis of JEV NS2B(H)- Den NS3p at different time. But no signals of expected protein

Slide 33: Plasmid digestion to check insert. JEV NS2B(H)- Den NS3p stock JEV NS2B(H)- Den NS3p pTrc plasmid check linearize insert 23.13 kb 9.42 kb 6.56 kb 4.56 kb 2.32 kb 2.03 kb

Slide 34: 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.

Slide 35: 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.

Slide 36: Future work Purification of the recombinant protein Protease activity assays, the comparison of protease activity to hybrids protein and dengue NS2B(H)/NS3p

Slide 37: NS2B(H) NS2B(H) C-terminal NS3p RR = 7.029 kDa RK = 20.309 kDa RQ = 28.884 kDa

Slide 38: Blast conferm