Refolding Record:
| Protein | |
|---|---|
| Protein Name | Complement regulatory protein Crry |
| Abbreviated Name | Crry |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Rat |
| UniProt Accession | Q63135 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 525 |
| Molecular Weight | 58030.9 |
| Pi | 5.21 |
| Molecular Weight | 58030.9 |
| Disulphides | >10 |
| Full Sequence |
GQCPAPPLFPYAKPINPTDESTFPVGTSLKYECRPGYIKRQFSITCEVNSVWTSPQDVCIRKQCETPLDPQNGIVHVNTDIRFGSSITYTCNEGYRLIGSSSAMCIISDQSVAWDAEAPICESIPCEIPPSIPNGDFFSPNREDFHYGMVVTYQCNTDARGKKLFNLVGEPSIHCTSIDGQVGVWSGPPPQCIELNKCTPPHVEN
AVIVSKNKSLFSLRDMVEFRCQDGFMMKGDSSVYCRSLNRWEPQLPSCFKVKSCGAFLGELPNGHVFVPQNLQLGAKVTFVCNTGYQLKGNSSSHCVLDGVESIWNSSVPVCEQVICKLPQDMSGFQKGLQMKKDYYYGDNVALECEDGYTLEGSSQSQCQSDASWDPPLPKCVSQVICKLPQDMSGFQKGLQMKKDYYYGDNVALECEDGYTLEGSSQSQCQSDASWDPPLPKCVSRSNSGLIAGIFIGIIVLILFIIFSYWMIMKFKKRNSTNEKCKEVGIYLNSKEDSCVQPQSLLTSQENNSTSSPARNSLTQEV
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Fraser DA, Harris CL, Smith RA, Morgan BP. (2002) Protein Science, 11, 2512-21 |
| Project Aim | Over expression & Renaturation |
| 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 h |
| Expression Vector | pET26b |
| Expression Protocol | Bacterial expression of sCrry-Cys by fermentation High-bacterial culture densities were obtained by fermentation of E. coli in a Bioflo 3000 Bioreactor (New Brunswick Scientific) with a 2-L bioreactor culture vessel. The fermenter was prepared according to the manufacturer\'s instructions. Briefly, the temperature was set at 37°C for the duration of the fermentation, and the pH was monitored. Maximum values for agitation and airflow were set to 750 rpm and 2 L/min, respectively. The vessel, containing 2 L of sterile NZCYM medium (Sigma) supplemented with kanamycin (50 µg/mL) and 0.1% polyethylene glycol (anti-foaming agent), was then inoculated with 20 mL of an overnight culture from a single colony of chemically competent E. coli BL21(DE3) transformed with the bacterial expression vector pET26b containing sCrry-Cys cDNA. The Bioflo 3000 was set to monitor the amount of dissolved O2 within the culture and automatically adjust agitation and airflow within the set maximum values to maintain a dissolved O2 content of 50% for maximum bacterial growth density. Cultures were grown for 4 h until the bacteria were in their log phase of growth (OD600 = 5–8). Protein expression was then induced by adding sterile filtered isopropyl ß D thiogalactopyranoside (IPTG) to a final concentration of 1 mM. The fermentation culture was harvested at 3 h post induction, and centrifuged at 10,000g for 10 min. Cell pellets were stored at -40°C. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 5-8 = 600 |
| Cell Disruption Method | High pressure homogenization |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 5 mL of 50 mM Tris, 1 mM EDTA, and 50 mM NaCl (pH 8.0) |
| Solubilization Buffer | 8 M urea, 20 mM Tris, 1 mM EDTA, and 50 mM 2-mercaptoethanol at pH 8.5 |
| Refolding Buffer | 0.02 M ethanolamine, 1 mM EDTA (pH 11.0) |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 11.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 24 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | A frozen cell pellet from 2 L of culture was thawed at 37°C in a shaker-incubator and washed twice in 5 mL of 50 mM Tris, 1 mM EDTA, and 50 mM NaCl (pH 8.0) per gram of pellet. The pellet was then resuspended in 5 mL of ice-cold wash buffer per gram pellet and blended using an electric blender until only fine particles remained. The cells were then disrupted by two passes at 12,000 Psi through an Emulsiflex C5 High Pressure Homogenizer (Glen Creston) at 4°C. The homogenized pellet was immediately centrifuged at 10,000g for 10 min, and the supernatant discarded. The pellet was washed three times in ice-cold wash buffer. Inclusion bodies were solubilized by resuspension of the pellet to 5 mg/mL in equilibration buffer (8 M urea, 20 mM Tris, 1 mM EDTA, and 50 mM 2-mercaptoethanol at pH 8.5) and refolded using a method based on that of Dodd et al. (1995). Solubilized sCrry-Cys inclusion bodies were refolded by rapid dilution 1/40 into cold 0.02 M ethanolamine, 1 mM EDTA (pH 11.0), and left static for 24 h at 4°C. The solution was then concentrated by ultrafiltration to a final volume of 750 mL and buffer exchanged into PBS. Soluble recombinant Crry was purified by anion exchange. Protein was loaded onto a Source 15Q column (Amersham Pharmacia) in 20 mM Tris (pH 8.9), and eluted with a linear gradient to 1 M NaCl, 20 mM Tris (pH 8.9), over 30 column volumes. Protein was run on SDS-PAGE to confirm purity and analyzed by MALDI-Tof (Bruker Relex 3 spectrometer; Bruker UK Ltd.) to confirm molecular mass. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |