Refolding Record:
| Protein | |
|---|---|
| Protein Name | Prothiolsubtilizin E |
| Abbreviated Name | n/a |
| SCOP Family | Subtilases |
| Structure Notes | |
| Organism | Bacillus subtilis |
| UniProt Accession | P04189 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha/Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 276 |
| Molecular Weight | 27730.8 |
| Pi | 6.3 |
| Molecular Weight | 27730.8 |
| Disulphides | Unknown |
| Full Sequence |
AQSVPYGISQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLNVRGGASFVPSETNPYQDGSSHGTHVAGTIAALNNSIGVLGVSPSASLYAVKVLDSTGSGQYSWIINGIEWAICNNMDVINMSLGGPTGSTALKTVVDKAVSSGIVVAAAAGNEGSSGSTSTVGYPAKYPSTIAVGAVNSSNQRASFSSAGSELDVMAPGVSIQSTLPGGTYGAYNGTSMATPHVAGAAALILSKHPTWTNAQVRDRLESTATYLGNSFYYGKGLINVQAAAQ
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Li Y, Inouye M. (1994) J Biol Chem, 269, 4169-4174 |
| Project Aim | Folding |
| 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 | 3-4 h |
| Expression Vector | pET11a |
| Expression Protocol | Protein Expression and Purification-The plasmid pETlla-rothiol- subtilisin was first established in E. coli strain C183 (20) and then transferred to T7 expression host strain BL2UDE3) (18). The expression of prothiolsubtilisin was performed at 37 \"C in M9 medium containing ampicillin (200 pg/mU supplemented with 2% casamino acids. At a Klett reading of 50 with a red filter, IPTG was added to the culture medium to a final concentration of 1 m to induce gene expression. After 3-4 h of induction, the cells were harvested by centrifugation (5000 X g, 10 min). Harvested cells from a 1-liter culture were washed once with ice-cold 25 m Tris-HC1 buffer (pH 7:O) containing 150 m NaCl. The cells were then suspended in the same buffer containing 1 m EDTA, 1 m phenylmethanesulfonyl fluoride, and 0.03 mg/ml DNase I and sub sequently disintegrated by a French press (twice at 750 psi). The disrupted cells were centrifuged at 10,000 x g for 10 min. Prothiolsubtilisin produced as inclusion bodies was recovered in the pellet. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 25 m Tris-HC1 buffer (pH 7:O) containing 150 m NaCl |
| Solubilization Buffer | 15 ml of 6 M guanidine HCl in |
| Refolding Buffer | 10 m Tris-HC1 (pH 7.0) containing 0.5 M (NH,),SO,, 1 m CaClZ, 5 m B-mercaptoethanol, and a variable amount of urea |
| Pre-Refolding Purification | Size-exclusion chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 7.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 6 h |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 5 mM |
| Refolding Protocol | The inclusion bodies were then solubilized in 15 ml of 6 M guanidine HCl in H2O. After overnight incubation at 4 \"C, the insoluble materials were removed by centrifugation at 90,000 x g for 40 min. The supernatantase was then dialyzed overnight against an excess of 50 m sodium-potassium phosphate buffer (pH 5.0) containing 5 M urea at 4 \"C. The dialysate was centrifuged at 90,000 xg for 40 min, and prothiolsubtilisin was recovered in the supernatant. The purification of prothiolsubtilisin was camed out as follows. The supernatant (15 ml) was applied to a cation-exchange column (1.5 x 15 cm) of CM-Sephadex C-50 equilibrated with 50 m sodium-potassiumphosphate buffer (pH 5.0), 5 M urea. After the column was washed with 2 column volumes of equilibration buffer, a 0-0.6 M NaCl gradient elution was camed out. Prothiolsubtilisin was eluted from the column between 0.15 and 0.2 M NaCI. The yield of purified prothiolsubtilisin was estimated to be 40-50 mgAiter of culture. In Vitro Renaturation ofProthiolsubtilisin-Renaturation of prothiolsubtilisin puritied from the CM-Sepharose column was performed by a stepwise dialysis procedure against 10 m Tris-HC1 (pH 7.0) containing 0.5 M (NH,),SO,, 1 m CaClZ, 5 m p-mercaptoethanol, and a variable amount of urea. The protein sample was first dialyzed against 100 volumes of buffer containing 4 M urea at 4 \"C for 2 h. Then, the initial buffer was sequentially replaced every 2 h with buffer containing decreasing amounts of urea from 2 to l M and then to 0.5 M. Finally, urea was completely removed from the buffer. After removing precipitates by centrifugation at 90,000 x g for 40 min, the final supernatant was applied to a Sephacryl S-200HR column (2.5 x 114 cm) equilibrated with the final dialysis buffer. The purified product was a complex consisting of the propeptide and the mature subtilisin sequence. To separate them, 15 ml of the complex fraction from the Sephacryl S-2OOHR column was dialyzed against 100 volumes of 50 m sodium-potassium phosphate buffer (pH 5.0). The dialysate was then concentrated to 4 ml by an Amicon ultrafiltration system using a Y\"3 membrane (Diaflo, Amicon Corp.). The concentrated solution was applied to a Pharmacia LKE! Biotechnology CM-Sepharose Fast Flow column (1 x 10 cm) on a fast protein liquid chromatography system Pharmacia). |
| Refolding Assay | Circular Dichroism (uv-CD) |
| Refolding Chaperones | None |
| Refolding Additives | (NH4)2SO4 |
| Additives Concentration | 0.5 M |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |