Refolding Record:
| Protein | |
|---|---|
| Protein Name | Secreted luciferase |
| Abbreviated Name | Luc |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Metridia longa |
| UniProt Accession | Q6UQE3 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 202 |
| Molecular Weight | 22020.7 |
| Pi | 7.61 |
| Molecular Weight | 22020.7 |
| Disulphides | Unknown |
| Full Sequence |
KSTEFDPNIDIVGLEGKFGITNLETDLFTIWETMEVMIKADIADTDRASNFVATETDANRGKMPGKKLPLAVIMEMEANAFKAGCTRGCLICLSKIKCTAKMKVYIPGRCHDYGGDKKTGQAGIVGAIVDIPEISGFKEMAPMEQFIAQVDRCASCTTGCLKGLANVKCSELLKKWLPDRCASFADKIQKEVHNIKGMAGDR
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Borisova VV, Frank LA, Markova SV, Burakova LP, Vysotski ES. (2008) Photochem Photobiol Sci., 7, 1025-31 |
| Project Aim | Over expression & Renaturation |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | E coli |
| Expression Strain | BL21 CodonPlus (DE3)-RIPL |
| Expression Temp | 37.0 |
| Expression Time | 3 h |
| Expression Vector | pET22b+ |
| Expression Protocol | The E. coli cells BL21 CodonPlus (DE3)-RIPL (Stratagene) transformed with the plasmids pET22-MLuc164 and pET22-MLuc39 were cultivated with vigorous shaking at 37 ◦ C in LB medium containing 200 lg mL−1 ampicillin. When the culture reached an OD600 of 0.8–1.0, the luciferase synthesis was induced with 100 mg L−1 IPTG. After induction, the cultivation was continued for 3 h. Cells were harvested by centrifugation at 4 ◦ C. Cell paste was resuspended (1 : 5, w/v) in buffer A (20 mM Tris–HCl pH 7.0) disrupted by sonication (20 s ×6) at 0 ◦ C and centrifuged thereafter. The pellet was sequentially washed with 0.15 M NaCl in buffer A, 0.5% Tween 20 in buffer A ( ×3), and buffer A. All the washing procedures were followed by centrifugation at 4 ◦ C. The final pellet was extracted either with 6 M urea in buffer B (50 mM Tris–HCl pH 8.8), or 6 M guanidine HCl. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.8-1.0 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | gel filtration |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 0.15 M NaCl, 20 mM Tris–HCl pH 7.0, 0.5% Tween 20 |
| Solubilization Buffer | 50 mM Tris–HCl pH 8.8, 6 M urea |
| Refolding Buffer | 5 mM cysteine, 0.5 mM cystine, 50 mM Tris–HCl pH 8.8 |
| Pre-Refolding Purification | gel filtration |
| Tag Cleaved | no tag |
| Refolding pH | 8.8 |
| Refolding Temperature | 8.0 |
| Protein Concentration | n/a |
| Refolding Time | 12 h |
| Redox Agent | Cysteine/Cystine |
| Redox Agent Concentration | 5/0.5 mM |
| Refolding Protocol | The inclusion bodies were dissolved in 6 M guanidine HCl, thereafter the equal aliquots were put (by drops) into refolding solutions in the ratio of 1 : 100, stored at 8 ◦ C and bioluminescence was monitored. Refolding solution compositions are as follows: No. 1 - 1 mM GSH, 0.5 mM GSSG in buffer B; No. 2 - 5 mM GSH, 0.5 mM GSSG in buffer B; No. 3 - 0.5 mM GSH, 5 mM GSSG in buffer B; No. 4 - 5 mM cysteine, 0.5 mM cystine in buffer B; No. 5 - buffer B; No. 6 - buffer A; No. 7 - 20 mM Bis-tris propane HCl pH 6.0; No. 8 - 100 mM NaCl in buffer B; No. 9 - 1 M NaCl in buffer B; No. 10 - 0.1% Tween 20 in buffer B; No. 11 - 1% Tween 20 in buffer B, No. 12 - 10% glycerol in buffer B; No. 13 - 0.1% gelatine in buffer B; No. 14 - 0.01% gelatine in buffer B; No. 15 - 5 mM GSH, 0.5 mM GSSG, 0.1% Tween 20 in buffer B; No. 16 - 5 mM GSH, 0.5 mM GSSG, 0.1 M NaCl in buffer B. The luciferase activity was measured with a BLM 8801 luminometer (SKTB “Nauka”, Russia) by injection of 5 lL of 10 lM coelenterazine in methanol into cuvette containing 5 lL luciferase sample and 500 lL SM–buffer (50 mM Tris–HCl pH 7.5, 0.1 M NaCl, 10 mM MgSO4 , 0.01% gelatine). In 12 h the samples were concentrated and analyzed by native PAGE. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 5–10 mg per 1 |
| Purity | n/a |
| Notes | n/a |