Refolding Record:
| Protein | |
|---|---|
| Protein Name | Hepatitis C virus core protein |
| Abbreviated Name | HCcAg |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Hepatitis C virus |
| UniProt Accession | Q99AU2 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | 339 AA N terminal Sequence containing Core Protein, and 43 AA of E1 Ag |
| Chain Length | 340 |
| Molecular Weight | 37305.0 |
| Pi | 10.2 |
| Molecular Weight | 37305.0 |
| Disulphides | 0 |
| Full Sequence |
MSTNPKPQRKTKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTSERSQPRGRRQPIPKARRPEGRTWAQPGYPWPLYGNEGMGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGCSFSIFLLALLSCLTIPASAYEVRNVSGIYHVTNDCSNSSIVYEAADMIMHTPGCVPCVRESNFSRCWVALTPTLAARNSSIPTTTIRRHVDLLVGAAALCSAMYVGDLCGSVFLVSQLFTFSPRRYETVQDCNCSIYPGHVSGHRMAWDMMMNWSPTTALVVSQLLRI
|
| Notes | UniProt Reference refers to HCV genotype 1b. The sequence used spands residues 1 to 339. Expression plasmid, pNAO.COE1.339 (for which the sequence could not be obtained) was digested with Sst1 and SalI and the 339 AA sequence of the HCV genome (genotype 1b) was inserted. The Sequence above refers to the 339 residue sequence |
| Expression | |
|---|---|
| Report | Acosta-Rivero, N., Rodriguez, A., Musacchio, A., Falcon, V., Suarez, V.M., Martinez, G., Guerra, I., Paz-Lago, D., Morera, Y., de la Rosa, M., Morales-Grillo, J., Duenas-Carrera, S., (2004) Biochem Biophys Res Commun., 325, 68-74 |
| Project Aim | Functional Studies,Assembly |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Pichia pastoris |
| Expression Strain | MP-36/C-E1.339 |
| Expression Temp | 37.0 |
| Expression Time | 24H |
| Expression Vector | pNAO.COE1.339 |
| Expression Protocol | Cells were transformed using electroporation and cultured in a 5L fermentor. A flow of induction flu |
| Method of Induction | Methanol |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Glass Beads |
| Lytic Agent | None |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 8 M urea in TEN buffer +/- 5 mM DTT |
| Refolding Buffer | TEN buffer, 1% glycerol, +/- 5 mM DTT |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Tag Cleaved | no |
| Refolding pH | 7.4 |
| Refolding Temperature | 37.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | DTT |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Pellet was treated with solubilisation buffer and sonificated before overnight incubation at 4degC. Resulting suspension was centrifuged(12 000g, 20 min). The extracted HCcAg was applied to cation-exchange column previously equilibrated with solubilisation buffer (with 1 mM DTT). Fractions containing HCcAg with more than 50% purity were pooled and run on reverse-phase HPLC column and eluted with 0.1 trifluoroacetic acid in water to 0.1% TFA in acetonitrile. Eluted HCcAg was dialysed against Refolding buffer. |
| Refolding Assay | Complex Assembly |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |