Refolding Record:
| Protein | |
|---|---|
| Protein Name | Oncoprotein E7 |
| Abbreviated Name | HPV-E7 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human papillomavirus |
| UniProt Accession | Q6YNY5 |
| 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 | 103 |
| Molecular Weight | 11699.4 |
| Pi | 4.46 |
| Molecular Weight | 11699.4 |
| Disulphides | Unknown |
| Full Sequence |
MIGKEVTVQDIILELSEVQPEVLPVDLFCEEELPNEQETEEEPDIERISYKVIAPCGCRHCEVKLRIFVHATEFGIRAFQQLLTGDLQLLCPDCRGNCKHDGS
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Liu X, Clements A, Zhao K, Marmorstein R. (2006) Biologycal Chemistry, 281, 578-586 |
| Project Aim | Drug Studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 3 |
| Expression Vector | pRSET |
| Expression Protocol | DNA fragments encoding residues 44–93 of native type 1A HPV E7 (HPV 1A E7-(44–93)) protein preceded by DNA encoding an MK sequence was cloned into the pRSET A vector for protein overexpression in Escherichia coli cells BL21 (DE3) (Invitrogen). Cells were grown in LB medium supplemented with 100 µg/ml ampicillin at 37 °C, and when the A595 nm reached 0.5, 1 mM isopropyl 1-thio--D-galactopyranoside and 100 µM zinc acetate were added to the media, and the cells were grown for 3 additional hours before they were harvested by centrifugation at 4000 x g for 20 min to isolate the cell pellet that was stored at -70 °C before protein purification |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.5 = 595 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 20 mM Tris-HCl, pH 7.5, 100 mM sodium chloride, 10 µM zinc acetate, and 10 mM dithiothreitol |
| Solubilization Buffer | 20 mM Ches, pH 10.15, 100 mM sodium chloride, 50 µM zinc acetate, 10 mM dithiothreitol, and 6 M guanidine-HCl denaturant |
| Refolding Buffer | 20 mM Ches, pH 10.15, 100 mM sodium chloride, 50 µM zinc acetate, 10 mM dithiothreitol |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 10.15 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | overnight |
| Redox Agent | DTT |
| Redox Agent Concentration | 10 mM |
| Refolding Protocol | Frozen cell pellets were suspended in 20 mM Tris-HCl, pH 7.5, 100 mM sodium chloride, 10 µM zinc acetate, and 10 mM dithiothreitol and lysed by sonication. The cell lysates were centrifuged at 40,000 x g for 30 min, and the insoluble fraction containing the HPV 1AE7-(44–93) was solubilized in 20 mM Ches, pH 10.15, 100 mM sodium chloride, 50 µM zinc acetate, 10 mM dithiothreitol, and 6 M guanidine-HCl denaturant. This HPV 1AE7-(44–93) denaturant solution was dialyzed against the same buffer without guanidinium-HCl overnight followed by a 4-h dialysis against a buffer containing 20 mM Tris-HCl, pH 7.5, 50 mM sodium chloride, 10 µM zinc acetate, and 10 mM dithiothreitol. The solution containing the refolded HPV 1AE7-(44–93) protein was centrifuged to remove precipitates formed during the refolding process, and the supernatant was then applied to a pre-equilibrated ion exchange Q-Sepharose column (Amersham Biosciences). The flow-through from the Q-Sepharose column, harboring the HPV 1AE7-(44–93) protein, was collected, concentrated (Millipore), and loaded onto a Superdex 200 gel filtration column (Amersham Biosciences). Peak fractions containing HPV 1AE7-(44–93) were judged to be greater than 95% pure by SDS-PAGE analysis with a typical yield of 100 mg of recombinant protein from 12 liters of growth culture. For the preparation of selenium-derivatized protein, base substitution encoding a single L76M mutation was introduced into the above construct by mutagenesis (QuikChange mutagenesis kit, Stratagene), and protein overexpression was carried out in the methionine auxotrophic E. coli cells B834 (DE3) (Novagen). B834 (DE3) cells were grown in selenomethionine-containing minimal media supplemented with the other 19 amino acids (35). Selenomethionine-derivatized protein was purified essentially as described for the wild-type construct with similar yield. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 95% |
| Purity | n/a |
| Notes | n/a |