Refolding Record:
| Protein | |
|---|---|
| Protein Name | Rhizopuspepsin |
| Abbreviated Name | n/a |
| SCOP Family | Pepsin Like |
| Structure Notes | |
| Organism | Rhizopus chinensis |
| UniProt Accession | P06026 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 325 |
| Molecular Weight | 34239.1 |
| Pi | 4.6 |
| Molecular Weight | 34239.1 |
| Disulphides | Unknown |
| Full Sequence |
AGVGTVPMTDYGNDVEYYGQVTIGTPGKKFNLDFDTGSSDLWIASTLCTNCGSRQTKYDPKQSSTYQADGRTWSISYGDGSSASGILAKDNVNLGGLLIKGQTIELAKREAASFANGPNDGLLGLGFDTITTVRGVKTPMDNLISQGLISRPIFGVYLGKASNGGGGEYIFGGYDSTKFKGSLTTVPIDNSRGWWGITVDRATVGTSTVASSFDGILDTGTTLLILPNNVAASVARAYGASDNGDGTYTISCDTSRFKPLVFSINGASFQVSPDSLVFEEYQGQCIAGFGYGNFDFAIIGDTFLKNNYVVFNQGVPEVQIAPVAQ
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Flentke GR, Glinski J, Satyshur K, Rich DH. (1999) Protein Expression and Purification, 16, 213-220 |
| Project Aim | Purification & characterization |
| Fusion | N-terminal polyhistidine peptide tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3)pLysS |
| Expression Temp | 37.0 |
| Expression Time | 4 h |
| Expression Vector | pET16b |
| Expression Protocol | The rhizopuspepsinogen gene was excised from pET3a-RPN with bpu1102I and NdeI. The gene was ligated into a bpu1102I, NdeI digested pET16b plasmid (Novagen), and transformed into Escherichia coli strain DH5-a. The resulting plasmid, pET16b-RPN, was isolated, confirmed by restriction analysis, and transformed into the E. coli strain BL21(DE3)plysS cells. The pET16b-RPN/Bl21(DE3)plysS cells were grown overnight at 37°C with shaking in 5-ml cultures of Luria broth that contained 30 mg/ml of chloramphenicol and 50 mg/ml of ampicillin. One milliliter of the overnight culture was used to inoculate 500 ml of Luria broth containing 30 mg/ml chloramphenicol and 100 􏰃g/ml ampicillin. This culture was grown at 37°C with shaking. When the absorbance at 600 nm reached 0.9 – 1.0, IPTG was added to obtain a final concentration of 1.25 mM. After incubation for an additional 4 h, the cells were harvested by centrifugation at 5000g and resuspended in buffer (50 mM Tris–HCl, 150 mM NaCl, 1 mM MgCl 2, pH 7.4) at a concentration of 5 ml/g wet cells. The resulting slurry was stored at-20°C. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.9-1.0 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 8 M urea, 50 mM CAPS, 1 mM EDTA, 1 mM glycine, 500 mM NaCl, 300 mM B-mercaptoethanol, pH 10.5 |
| Refolding Buffer | Different refolding buffers please see the refolding notes |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no |
| Refolding pH | 0.0 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Inclusion body proteins were refolded by using the protocol of Lowther et al. (12) that was systematically modified to achieve higher yields of folded protein. Inclusion bodies were dissolved to a concentration of 1 mg inclusion bodies per milliliter of freshly deionized denaturation buffer (8 M urea, 50 mM CAPS, 1 mM EDTA, 1 mM glycine, 500 mM NaCl, 300 mM B-mer-captoethanol, pH 10.5). After stirring for 1 h at room temperature, the denatured protein was dialyzed against 5 vol of 50 mM Tris–HCl, pH 11. After 1 h of dialysis the buffer was exchanged with fresh 50 mM Tris–HCl, pH 11, and dialyzed for an additional 1 h at room temperature. Dialysis was continued for 24 h in 5 vol of 50 mM Tris–HCl, pH 7.5, at 4°C. The final dialysis was stirred overnight against 5 vol of 50 mM MOPS, 300 mM NaCl, pH 7.0. The resulting refolded protein solution was centrifuged at 24,000g for 30 min to remove insoluble material. Triton X-100 was added to obtain a final concentration of 0.05% (v/v). The protein solution was concentrated via tangential-flow ultrafiltration (PCBCOMP04 membrane, Millipore) foll owed by pressure ul trafil trati on usi ng Mi l l i pore YM-30 membranes. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | Refolding buffers 1. 50 mM Tris-Hcl, pH 11 room temperature 1 h (2 times) 2. 50 mM Tris-Hcl, pH 7.5 at 4c 24 h 3. 50 mM MOPS, 300 mM NaCl, pH 7.0 over night |