Refolding Record:
| Protein | |
|---|---|
| Protein Name | UL82as |
| Abbreviated Name | UL82as |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human cytomegalovirus |
| UniProt Accession | n/a |
| 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 | 154 |
| Molecular Weight | 16339.5 |
| Pi | 11.5 |
| Molecular Weight | 16339.5 |
| Disulphides | Unknown |
| Full Sequence |
MTSVRAPLLPLRRLCPVRISAGDSPAWVSESSSPLASSKPANNASDRGVGVGVEERSSSSSSSSSSSSSSVGGNPGDCGRNSHTAPRMTLLRGKRPARSCTWGRKGLILSGLPGVRVQHPRRKKWMRPSGCRCSKGKPIPNPLLGLDSHHHHHH
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Bego, M., Maciejewski, J., Khaiboullina, S., Pari. G., St Jeor, S. (2005) J Virol, 79, 11022-11034 |
| Project Aim | Functional Studies |
| Fusion | C-terminal hexahis +V6 epitope |
| 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 | 24h |
| Expression Vector | pET102-TOPO |
| Expression Protocol | Induced cells were harvested by centrifugation. Bacterial pellets were re-suspended in re-suspension buffer(20mM TrisHCl, pH 8.0), 4ml per 100ml of initial culture. Protease inhibitors were added and then cells lysed by three cycles of freeze/thaw using dry ice and methanol mix and a 37degC water bath. Cells were further lysed using an 18-gauge syringe between cycles. Cells were centrifuged(10min, 4degC, 10000f) and pellets re-suspended in cold isolation buffer (2M urea, 20mM TrisHCl, 0.5 M NaCl, 2% Triton X-100, pH 8.0). Freeze/thaw procedure was then repeated and followed again by centrifugation. The pellet was washed in isolation buffer, without urea, and resuspended in binding buffer(6M GuHCl, 20mM TrisHCl, 0.5M NaCl, 5mM imisazole, 1mM 2-mercaptoethanol, pH 8.0) using 5ml buffer for every 100ml culutre. The samples were then incubated at room temp. for 60min with gentle rocking. The samples were then centrifuged(15min, 4degC). Supernatant was filtrated using 0.45 microm filters inorder to remove remaining particles. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Freeze-thaw |
| Lytic Agent | Detergents |
| Pre-Refolding Purification | Affinity chromotography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Nickel-chelating chromatography |
| Wash Buffer | n/a |
| Solubilization Buffer | 6 M urea, 20 mM TrisHCl, 0.5 M NaCl, 20 mM imidazole, 1 mM 2-mercaptoethanol, pH 8.0 |
| Refolding Buffer | 20 mM TrisHCl, 0.5 M NaCl, 20 mM imidazole, 1 mM 2-mercaptoethanol, pH 8.0 |
| Pre-Refolding Purification | Affinity chromotography |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 1 mM |
| Refolding Protocol | HiTrap Chelating column were washed with 5ml of distilled water, prior to loading with 0.5 ml, 0.1 M NiSO4. 5-10ml binding buffer was then used to equilibrate the column and the sample was loaded. The columns were then washed with 10ml binding buffer followed by 10ml solubilization buffer. The proteins were then refolded in the column using a linear 6-0 M urea gradient. The refolded protein was eluted using 0-1mM 2-mercaptoethanol linear gradient. |
| Refolding Assay | SDS-PAGE,Sequence Analysis |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |