Refolding Record:
| Protein | |
|---|---|
| Protein Name | Outer membrane protein C |
| Abbreviated Name | OmpC |
| SCOP Family | Porin |
| Structure Notes | |
| Organism | Salmonella typhi Ty21a |
| UniProt Accession | P0A263 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Membrane and cell surface proteins and peptides |
| Molecularity | Trimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 357 |
| Molecular Weight | 39238.4 |
| Pi | 4.5247 |
| Molecular Weight | 39238.4 |
| Disulphides | Unknown |
| Full Sequence |
MEIYNKDGN KLDLFGKVDG LHYFSDDKGS DGDQTYMRIG FKGETQVNDQ LTGYGQWEYQ IQGNQTEGSN DSWTRVAFAG LKFADAGSFD YGRNYGVTYD VTSWTDVLPE FGGDTYGADN FMQQRGNGYA TYRNTDFFGL VDGLDFALQY QGKNGSVSGE NTNGRSLLNQ NGDGYGGSLT YAIGEGFSVG GAITTSKRTA DQNNTANARL YGNGDRATVY TGGLKYDANN IYLAAQYSQT YNATRFGTSN GSNPSTSYGF ANKAQNFEVV AQYQFDFGLR PSVAYLQSKG KDISNGYGAS YGDQDIVKYV DVGATYYFNK NMSTYVDYKI NLLDKNDFTR DAGINTDDIV ALGLVYQF
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Kumar P.D., Krishnaswamy, S. (2005) Protein Expression and Purification, 40, 126-133 |
| Project Aim | Structural Studies |
| 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 | 5 hours |
| Expression Vector | pLF2 |
| Expression Protocol | Escherichia coli BL21 (DE3) was transformed with pLF2. BL21/pLF2 was grown overnight in LB medium. One percent of this inoculum was added to 1 L of LB medium and the culture was grown in a shaker at 37 °C. At 0.6 OD, culture was induced with 0.2 mM IPTG and kept for expression for 5 h. Cell pellet was obtained by centrifuging at 7000 rpm for 13 min at 20 °C. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.6 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50 mM Tris, pH 8.5, 0.1 M NaCl, and 2% Triton X-100 |
| Solubilization Buffer | 50 mM Tris, pH 8.5, 0.1 M NaCl, and 4 M urea |
| Refolding Buffer | 50 mM Tris, pH 8.5, 0.1 M NaCl, 10% (v/v) glycerol, and 0.2% (v/v) polyoxyethylene-9-laurylether |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The cell pellet was washed twice with 0.8% (w/v) saline. Cells were disrupted by sonication (SONICS, Vibra cell 300 W, total run time of 3 min with pulsar of 9 s on and 5 s off). Crude IBs were obtained by centrifugation of cell lysate at 7000 rpm for 10 min at 20 °C (Fig. 1). IBs were further washed two times with TTN buffer (50 mM Tris, pH 8.5, 0.1 M NaCl, and 2% Triton X-100) and with TN buffer (50 mM Tris, pH 8.5, and 0.1 M NaCl). After each wash, the resuspended solution was centrifuged at 7000 rpm for 7 min at 20 °C. Inclusion bodies (IBs) were solubilized in Tris-Urea buffer (50 mM Tris, pH 8.5, 0.1 M NaCl, and 4 M urea). Solubilization was done for 5 h on moderate shaking at 37 °C. This suspension was centrifuged at 13,000 rpm for 20 min at 20 °C to obtain unfolded OmpC (UFOmpC) in the supernatant. UFOmpC was passed through 0.2 ìm filter to remove aggregates and particulate matter. Refolding buffer [50 mM Tris, pH 8.5, 0.1 M NaCl, 10% (v/v) glycerol, and 0.2% (v/v) polyoxyethylene-9-laurylether (C12E9, Sigma)] was kept at 4 °C with stirring, into which the UFOmpC was rapidly added in 1:5 protein to buffer ratio. The solution was incubated at 4 °C overnight and concentrated using ultrafiltration stirred cell (Amicon, Millipore) with 50 kDa MWCO membrane. The protein solution was subjected to ultracentrifugation at 30,000 rpm for 90 min at 20 °C using RPR 50-T rotor (Hitachi ultracentrifuge) to remove small aggregates and particulate matter. rfOmpC was further purified by ion-exchange chromatography. Q-Sepharose fast flow (Sigma) ion exchange column was equilibrated with binding buffer (50 mM Tris, pH 8.5, 0.1 M NaCl, 10% glycerol, and 0.2% C12E9). rfOmpC was loaded onto the column at 10 ml/h flow rate. Column was washed with the binding buffer (five bed volumes) to remove unbound protein and eluted with binding buffer containing 1 M NaCl. To reduce the salt concentration in the eluent to 0.1 M, the eluted protein was diluted with binding buffer without NaCl and concentrated, in a stepwise manner, in ultrafiltration stirred cell with 50 kDa MWCO membrane. |
| Refolding Assay | Far-UV Circular Dichroism |
| Refolding Chaperones | None |
| Refolding Additives | None,Glycerol |
| Additives Concentration | 10% |
| Refolding Yield | 0.9g/L |
| Purity | 90 |
| Notes | |