Refolding Record:
| Protein | |
|---|---|
| Protein Name | HPV16 E7-MS2 polymerase fusion |
| Abbreviated Name | HPV16 E7MS2 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human papillomavirus |
| UniProt Accession | P03129 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | E7(aa.9-98)-MS2 polymerase (aa.444-544) |
| Variants | n/a |
| Chain Length | 191 |
| Molecular Weight | 21321.0 |
| Pi | 5.1 |
| Molecular Weight | 21321.0 |
| Disulphides | Unknown |
| Full Sequence |
HE YMLDLQPETT DLYCYEQLND SSEEEDEIDG PAGQAEPDRA
HYNIVTFCCK CDSTLRLCVQ STHVDIRTLE DLLMGTLGIV CPICSQKP LAADYY VVSPPTAVSV YTKTPYGRLL ADTRTSGFRL ARIARERKFF SEKHDSGRYI AWFHTGGEIT DSMKSAGVRV IRTSEWLTPV PTFPQECGPA SSPR
|
| Notes | UniProt for MS2: RRPO_BPMS2 |
| Expression | |
|---|---|
| Report | Suttnar J, Dyr JE, Hamsikova E, Novak J, Vonka V (1994) J Chromatography B, 656, 123-126 |
| Project Aim | Structure-Function |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 37.0 |
| Expression Time | n/a |
| Expression Vector | n/a |
| Expression Protocol | no details provided |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 50mM Tris, 0.1% sodium desoxycholate, 0.02% lysozyme, 1mM EDTA, pH 7 |
| Solubilization Buffer | 1% SDS/8M urea/0.01M NaOH |
| Refolding Buffer | 50mM TrisHCl pH 8.0, 0.01% mercaptoethanol |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 7.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 10 micrograms/m |
| Refolding Time | |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 0.01% |
| Refolding Protocol | E.coli cells containing overexpressed protein were harvested by centrifugation. 1g of cell pellets was resuspended in 3ml of Buffer A (10mM Tris, 10mM NaCl, 10mM DTT, 1mM PMSF, pH 7.5) and lysed by sonication. Following centrifugation (30min, 15000g, 4degC), the pellet was resuspended in 5ml Wash buffer and incubated for 1h. The suspension was then was then centrifuged again and the pellet resuspended in 9ml buffer A. To solubilize the inclusion bodies, 1ml of resuspended inclusion bodies was slowly added to 10ml of stirred solubilization buffer and stirring was condinued for 1h. The solution was then centrifuged at 15000g for 40min at 12degC. The solubilized inclusion bodies were then refolded by dialysis in refolding buffer at concentrations of about 10micrograms/ml at 4degC. The dialysed solution was then centrifuged and the supernatant applied to a MonoQ column preequilibrated with refolding buffer. The column was washed with refolding buffer containing 1% DTT and at pH 7.5. Fractions were eluted at a flow rate of 1.0ml/min with a linear gradient from 0 to 0.5M NaCl in 30mi. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | 3 solubilization buffers tried - in 8M urea and 0.01M NaOH contaminating proteins remained insoluble, wherease in 1% SDS all proteins in IBs dissolved. Therefore, purification was much more effective using 8M urea or 0.01M NaOH |