Refolding Record:
| Protein | |
|---|---|
| Protein Name | Ribosome-inactivating protein gelonin |
| Abbreviated Name | GEL |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Gelonium multiflorum |
| UniProt Accession | P33186 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 251 |
| Molecular Weight | 28172.2 |
| Pi | 9.12 |
| Molecular Weight | 28172.2 |
| Disulphides | 1 |
| Full Sequence |
GLDTVSFSTKGATYITYVNFLNELRVKLKPEGNSHGIPLLRKKCDDPGKCFVLVALSNDNGQLAEIAIDVTSVYVVGYQVRNRSYFFKDAPDAAYEGLFKNTIKTRLHFGGSYPSLEGEKAYRETTDLGIEPLRIGIKKLDENAIDNYKPTEIASSLLVVIQMVSEAARFTFIENQIRNNFQQRIRPANNTISLENKWGKLSFQIRTSGANGMFSEAVELERANGKKYYVTAVDQVKPKIALLKFVDKDPK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Guo C, Li Z, Shi Y, Xu M, Wise JG, Trommer WE, Yuan J. (2004) Protein Expression and Purification, 37, 361-367 |
| Project Aim | Protein refolding |
| Fusion | C-terminal intein |
| 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 | 16 h |
| Expression Vector | pBSL-C |
| Expression Protocol | o fuse the target gene to the C-terminus of CBD-intein reading frame, the Gel gene (753 bp) was amplified from plasmid pUC-Gel by polymerase chain reaction (PCR) with the following primers which, respectively, contained unique restriction sites for AgeI (forward) and PstI (reverse) to facilitate cloning: forward primer: 5′-GGT GGT ACC GGT GGC CTG GAT ACC GTG AGC-3′, reverse primer: 5′-GGT GGT CTG CAG TTA TTT CGG ATC TTT ATC G AC-3′. The conditions used were: 95 °C for 5 min, 30 cycles of (94 °C 30 s, 52 °C 30 s, and 72 °C 1 min), and a final extension of 72 °C for 10 min. The PCR product was purified and digested with corresponding enzymes, subsequently inserted into the vector pBSL-C at AgeI and PstI sites of MCS to form the recombinant plasmid pBSL-Gel [14]. After restriction enzyme digestion and DNA sequencing verification, the pBSL-Gel was transformed into competent E. coli strain BL21(DE3) by calcium chloride method [13]. The resulting engineered strain E. coli BL21(DE3)/pBSL-Gel was grown in LB medium supplemented with 100 g/mL ampicillin at 37 °C until the optical density (OD) at 600 nm reached 0.5–0.6, and then induced by 0.5 mM IPTG at 12 °C for 16 h. The fusion expression product, CBD-intein–Gel was confirmed by SDS–PAGE and Western blot analysis. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.5-0.6 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Detergents |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 10 mM Tris–HCl, 500 mM NaCl, 1 mM PMSF, pH 8.5, and 0.1% Triton X-100,4 M urea |
| Solubilization Buffer | 10 mM Tris–HCl, 500 mM NaCl, pH 8.5, 8 M urea |
| Refolding Buffer | 10 mM Tris–HCl, 500 mM NaCl, pH 8.5, 0.2 mM GSH, 0.02 mM GSSG, and 0.5 M L-Arg |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no |
| Refolding pH | 8.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 24 h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 0.2/0.02 mM |
| Refolding Protocol | Refolding of CBD-intein–Gel aggregate, The cultured cells were harvested by centrifugation at 6000 rpm for 10 min and the cell pellets from 1 L IPTG induced culture were suspended and sonicated in lysis buffer A (10 mM Tris–HCl, 500 mM NaCl, 1 mM PMSF, pH 8.5, and 0.1% Triton X-100). The lysate was then centrifuged at 12,000 rpm for 30 min. Because the fusion protein almost completely existed in the form of IBs by 10% SDS–PAGE analysis, the precipitate was washed with buffer A containing 4 M urea and centrifuged at 12,000 rpm for 30 min. This step was repeated twice to remove the cell debris and other impurities. Then the IBs were dissolved in buffer B (10 mM Tris–HCl, 500 mM NaCl, pH 8.5) containing 8 M urea to reach a protein concentration of about 0.3 mg/mL. The dissolved precipitate was kept for 2–3 h at room temperature to completely solubilize the aggregate. After centrifugation at 12,000 rpm for 30 min, about 10 mL supernatant was step-wisely dialyzed against 1 L buffer B containing 4, 2, and 1 M urea sequentially at 4 °C for about 17–18 h each. Finally, the protein solution was dialyzed against 500 mL buffer B containing 0.2 mM GSH, 0.02 mM GSSG, and 0.5 M L-Arg for in vitro refolding at 4 °C for 24 h. After centrifugation at 12,000 rpm for 30 min, the supernatant was further purified in the next step. The in vitro refolding process was simultaneously monitored by size-exclusion HPLC equipped with a SW 300 gel column (7.8 × 300 mm) with 10 mM Tris buffer, pH 8.5, as the mobile phase at a flow rate of 1.0 mL/min, and absorbance at 280 nm was monitored with a UV detector. |
| Refolding Assay | Western Blot,ELISA |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 0.5 M |
| Refolding Yield | 69% |
| Purity | n/a |
| Notes | n/a |