Refolding Record:
| Protein | |
|---|---|
| Protein Name | ProDer p 1 |
| Abbreviated Name | ProDer p 1 |
| SCOP Family | Papain-like Cysteine Proteinases |
| Structure Notes | |
| Organism | Dermatophagoides pteronyssinus (House-dust mite) |
| UniProt Accession | Unknown |
| 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 | 302 |
| Molecular Weight | 34275.2 |
| Pi | 5.52 |
| Molecular Weight | 34275.2 |
| Disulphides | 3 |
| Full Sequence |
RPSSIKTFEEYKKAFNKSYATFEDEEAARKNFLESVKYVQSNGGAINHLSDLSLDEFKNRFLMSAEAFEHLKTQFDLNAETNACSINGNAPAEIDLRQMRTVTPIRMQGGCGSCWAFSGVAATESAYLAYRNQSLDLAEQELVDCASQHGCHGDTIPRGIEYIQHNGVVQESYYRYVAREQSCRRPNAQRFGISNYCQIYPPNVNKIREALAQTHSAIAVIIGIKDLDAFRHYDGRTIIQRDNGYQPNYHAVNIVGYSNAQGVDYWIVRNSWDTNWGDNGYGYFAANIDLMMIEEYPYVVIL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Unpublished (0) J Virol Methods, 0, 0 |
| Project Aim | Vaccine studies |
| Fusion | C-terminal hexahis + pro seq |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | E.coli |
| Expression Strain | SE1 E coli cells |
| Expression Temp | 37.0 |
| Expression Time | 2hrs |
| Expression Vector | pStaby1 |
| Expression Protocol | Recombinant ProDer p 1 (ProDer p 1 coli) was expressed at 37°C after addition of 1 mmol/L isopropyl-thiogalactoside (Duchefa, Haarlem, The Netherlands) for 2 hours at an OD600 nm of 0.6 to 0.8. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.6-0.8 |
| Cell Disruption Method | Cell disrupter |
| Lytic Agent | None |
| Pre-Refolding Purification | Ni-NTA column |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | TRIS buffer 50 mmol/L, pH 7.5, Triton X-100 1%, followed by three step wash to remove detergent |
| Solubilization Buffer | 40 mL TRIS buffer 50 mmol/L, 300 mmol/L NaCl, and 6mol/L urea, pH 7.5; O/N at 4C |
| Refolding Buffer | 3-step dialysis to remove urea (from 6 mol/L to 2 mol/L urea, from 2 mol/L to PBS + 500 mmol/L L-arginine, from PBS + 500 mmol/L L-arginine to PBS) |
| Pre-Refolding Purification | Ni-NTA column |
| Tag Cleaved | no |
| Refolding pH | 7.5 |
| Refolding Temperature | 24.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The induced bacteria were collected by centrifugation (15 minutes at 9500g), resuspended in 40 mL cold TRIS buffer 50 mmol/L, pH 7.5, supplemented with Aprotinin 1 mmol/L (Sigma, Bornem, Belgium) and 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride 1 mmol/L (MP Biomedicals Solon, Ohio), and lysed by 2 passages through a cell disrupter (Cell D; Constant, Low March, United Kingdom) at 1800 bars. The cell lysate was centrifuged for 20 minutes at 3000g to isolate the inclusion bodies. The pellet was washed with TRIS buffer 50 mmol/L, pH 7.5, Triton X-100 1%, followed by 3 washing steps to remove the detergent. ProDer p 1 was subsequently extracted overnight at 4°C with 40 mL TRIS buffer 50 mmol/L, 300 mmol/L NaCl, and 6 mol/L urea, pH 7.5. After ultracentrifugation (45 minutes, 149,000g), the supernatant of extraction was applied at 3 mL/min on a Ni2+ chelate high-performance column (2.6 × 6 cm; GE Healthcare Bio-Sciences, Uppsala, Sweden) equilibrated with the extraction buffer. The column was washed with the starting buffer. Protein elution proceeded by stepwise increasing imidazole concentration in the buffer (from 0 to 400 mmol/L). Fractions containing purified ProDer p 1 (elution with 200 mmol/L imidazole) were pooled. ProDer p 1 coli was renatured by a 3-step dialysis to remove urea (from 6 mol/L to 2 mol/L urea, from 2 mol/L to PBS + 500 mmol/L L-arginine, from PBS + 500 mmol/L L-arginine to PBS). The recombinant allergen was concentrated by ultrafiltration (regenerated cellulose ultrafiltration membranes, cutoff 10 kd; Millipore, Billerica, Mass) and stored at –20°C. |
| Refolding Assay | Immunoassay,ELISA,Activity assay |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 500 mmol/L |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |