Refolding Record:
| Protein | |
|---|---|
| Protein Name | Cytomegalovirus protease |
| Abbreviated Name | CMV PR |
| SCOP Family | Herpes virus serine proteinase, assemblin |
| Structure Notes | |
| Organism | Human cytomegalovirus |
| UniProt Accession | P16753 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 268 |
| Molecular Weight | 29433.9 |
| Pi | 5.98759 |
| Molecular Weight | 29433.9 |
| Disulphides | 0 |
| Full Sequence |
MTMDEQQSQA VAPVYVGGFL ARYDQSPDEA ELLLPRDVVE HWLHAQGQGQ PSLSVALPLN INHDDTAVVG HVAAMQSVRD GLFCLGCVTS PRFLEIVRRA SEKSELVSRG PVSPLQPDKV
VEFLSGSYAG LSLSSRRCDD VEAATSLSGS ETTPFKHVAL CSVGRRRGTL AVYGRDPEWV TQRFPDLTAA DRDGLRAQWQ RCGSTAVDAS GDPFRSDSYG LLGNSVDALY IRERLPKLRY
DKQLVGVTER ESYVKASVSP EAHHHHHH
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Tomasselli AG, Paddock DJ, Curry KA, Garlick RL, Leone JW, Lull JM, Mutchler VT, Baker CA, Cavey GS, Mathews WR, Shelly JA, Finzel BC, Baldwin ET, Wells PA, Tomich CC (1998) Protein Expression and Purification, 14, 343-352 |
| Project Aim | Structure-Function |
| Fusion | C-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | None |
| Expression Temp | 42.0 |
| Expression Time | 4-5h |
| Expression Vector | pET21a |
| Expression Protocol | Cells were grown at 30degC in LB medium until A550 reached 0.6-0.8. The cells were then quickly mixed with an equal volume of LB medium which had been prewarmed to 50degC and immediately placed in a 42degC shaker waterbath. After 4-5h cells were harvested by centrifugation. |
| Method of Induction | Temperature Shift |
| Cell Density at Induction | OD 550 = 0.6-0.8 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 8M GdnHCl pH 7.0 |
| Refolding Buffer | 0.1M Mops, 20% glycerol, 0.2M NaCl, 2mM DTT pH 7.5 |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | yes |
| Refolding pH | 7.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | |
| Refolding Time | 48-72h |
| Redox Agent | DTT |
| Redox Agent Concentration | 2mM |
| Refolding Protocol | Cells were suspended in 10mM TrisHCl pH 8.0, 1mM EDTA at a cell density of A550 about 20 and then sonicated. Cell extracts were centrifuged and the pellet retained. Inclusion bodies from 1.5L of cell culture were dissolved in 50ml solubilization buffer and stirred on ice for approximately 1h. After centrifugation, 16ml of 0.1M Na-phosphate, 0.01M Tris pH 8.0, 4mM 2-mercaptoethanol, 6M GdnHCl (Buffer A) was added to the supernatant and batch-mixed with 10ml Ni-NTA resin for approximately 1h. The resin was then poured into a column and washed with 10 column volumes of Buffer A, followed by 12-15 column volumnes of buffer B (0.1M Na-phosphate, 12mM imidazole, 4mM 2-mercaptoethanol, 4M GdnHCl pH 6.2). The protein was then eluted with a 40-column gradient from 1mM to 150mM imidazole in buffer C (0.1M Na-phosphate, 4mM 2-mercaptoethanol, 4M GdnHCl pH 6.8). Selected fractions were then pooled, then made up to 40% glycerol, 0.2M NaCl, 2mM DTT and then dialyzed against 2 x 4L of refolding buffer at room temperature for 48-72h for refolding and removal of the C-terminal autocleavable extension. |
| Refolding Assay | Amino acid sequencing |
| Refolding Chaperones | None |
| Refolding Additives | None,Glycerol |
| Additives Concentration | 20% |
| Refolding Yield | |
| Purity | |
| Notes | |