Refolding Record:
| Protein | |
|---|---|
| Protein Name | NS2B-NS3 Protease |
| Abbreviated Name | DEN2 NS2B-NS3 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Dengue virus type 2 |
| UniProt Accession | P29990 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 748 |
| Molecular Weight | 83277.4 |
| Pi | 6.19 |
| Molecular Weight | 83277.4 |
| Disulphides | Unknown |
| Full Sequence |
SWPLNEAIMAVGMVSILASSLLKNDIPMTGPLVAGGPLTVCYVLTGRSADLELERAADVKWEDQAEISGSSPILSITISEDGSMSIKNEEEEQTLTILIRTGLLVISGLFPVSIPITAAAWYLWEVKKQRAGVLWDVPSPPPMGKAELEDGAYRIKQKGILGYSQIGAGVYKEGTFHTMWHVTRGAVLMHKGKRIEPSWADVKKDLISYGGGWKLEGEWKEGEEVQVLALEPGKNPRAVQTKPGLFKTNAGTIGAVSLDFSPGTSGSPIIDKKGKVVGLYGNGVVTRSGAYVSAIAQTEKSIEDNPEIEDDIFRKRRLTIMDLHPGAGKTKRYLPAIVREAIKRGLRTLILAPTRVVAAEMEEALRGLPIRYQTPAIRAEHTGREIVDLMCHATFTMRLLSPVRVPNYNLIIMDEAHFTDPASIAARGYISTRVEMGEAAGIFMTATPPGSRDPFPQSNAPIIDEEREIPERSWNSGHEWVTDFKGKTVWFVPSIKAGNDIAACLSKNGKKVIQLSRKTFDSEYAKTRTNDWDFVVTTDISEMGANFKAERVIDPRRCMKPVILTDGEERVILAGPMPVTHSSAAQRRGRIGRNPKNENDQYIYMGEPLENDEDCAHWKEAKMLLDNINTPEGIIPSMFEPEREKVDAIDGEYRLRGEARTTFVDLMRRGDLPVWLAYRVAAEGINYADRRWCFDGVKNNQILEENVEVEIWTKEGERKKLKPRWLDARIYSDPLALKEFKEFAAGRK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Khumthong R, Angsuthanasombat C, Panyim S, Katzenmeier G. (2002) J Biochem Mol Biol., 35, 206-212 |
| Project Aim | Antigenic Determinant Identification |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | C41(DE3) |
| Expression Temp | 0.0 |
| Expression Time | 00 |
| Expression Vector | pTRCHisA |
| Expression Protocol | The recombinant NS2B-NS3(pro) and NS2B-NS3(pro)M proteins of the dengue virus type 2 were expressed as inclusion bodies in bacterial cells E. coli C41. They were purified by a modification of the method described previously (Champreda et al., 2000). |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 50mM Tris-Cl, pH 7.8, 1% (v/v) Triton X-100 |
| Solubilization Buffer | 100mM Tris-Cl, pH 7.8, 500mM NaCl, 6 M guanidinium hydrochloride |
| Refolding Buffer | 100mM Tris-HCl, pH 8.0, 300 mM NaCl |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 20 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The pellet fraction was washed 3 times with cold 50mM Tris-Cl, pH 7.8, 1% (v/v) Triton X-100 and solubilized by resuspension in 15ml of 100mM Tris-Cl, pH 7.8, 500mM NaCl, 6 M guanidinium hydrochloride. The denatured lysate was clarified by centrifugation at 4oC. The proteins were fractionated by passage through a HiTrap chelating Ni2+-affinity column (Pharmacia, Uppsala, Sweden) that contained 1ml of resin that was equilibrated with binding buffer A (20mM Tris-Cl, pH 7.8, 500mM NaCl, 6 M guanidinium hydrochloride). The column was washed with 10ml of a binding buffer, and was subsequently washed with 20ml of buffer B (buffer A with 50mM imidazole). The proteins were eluted from the column at a flow rate of 0.5ml min-1 in buffer C (100mM Tris-Cl, pH 7.8, 500mM NaCl, 6M guanidinium hydrochloride, 500mM imidazole). The fractions of 1ml were collected and the elution profile was monitored at A280. Fractions were analyzed by electrophoresis on 15% polyacrylamide gels. Peak fractions were pooled and applied to a Superdex 200 HR10/30 gel filtration column (Pharmacia). The column was eluted at a flow rate of 0.2ml min-1 with 24ml of an elution buffer (100mM Tris-Cl, pH 8.3, 300mM NaCl, 6 M guanidinium hydrochloride). Fractions of 0.5ml were collected and analyzed by SDS-PAGE. Elution fractions that contained the DEN proteins were pooled and the proteins were refolded by stepwise dialysis against 4 changes of a 200ml dialysis buffer (100mM Tris-HCl, pH 8.0, 300 mM NaCl) |
| Refolding Assay | HPLC |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |