Refolding Record:
| Protein | |
|---|---|
| Protein Name | 56kDa type-specific outer membrane antigen |
| Abbreviated Name | 56kDa protein |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Orientia tsutsugamushi/Rickettsia tsutsugamushi |
| UniProt Accession | P37915 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | aa 80-456 only |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 377 |
| Molecular Weight | 40897.3 |
| Pi | 6.26 |
| Molecular Weight | 40897.3 |
| Disulphides | Unknown |
| Full Sequence |
MTIAPGFRAEI GVMYLTNITA QVEEGKVKAD SVGETKADSV GGKDAPIRKR FKLTPPQPTI MPISIAVRDF GIDIPNQTSA ASTSRSLRLN DEQRAAARIA WLKNCAGIDY RVKNPNDPNG
PMVINPILLN IPQGNPNPVG NPPQRANPPA GFAIHNHEQW RHLVVGLAAL SNANKPSASP VKVLSDKITQ IYSDIKHLAD IAGIDVPDTS LPNSASVEQI QNKMQELNDL LEELRESFDG YLGGNAFANQ IQLNFVMPQQ AQQQGQGQQQ QAQATAQEAV AAAAVRLLNG NDQIAQLYKD LVKLQRHAGI KKAMEKLAAQ QEEDAKNQGE GDCKQQQGTS EKSKKGKDKE AEFDLSMIVG QVKLYADVMI TESVSI
|
| Notes | protein from O.tsutsugamushi Karp strain used |
| Expression | |
|---|---|
| Report | Ching W-M, Wang H, Eamsila C, Kelly DJ, Dasch GA (1998) Clin Diagn Lab Immunol, 5, 519-526 |
| Project Aim | Diagnostic studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 37.0 |
| Expression Time | overnight |
| Expression Vector | pET11a |
| Expression Protocol | Cells were grown in 2x TY borth, protein expression was induced with IPTG, cells were grown overnight. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 20mM TrisHCl pH 8.0, 2M urea |
| Solubilization Buffer | 20mM TrisHCl pH 8.0, 8M urea |
| Refolding Buffer | 20mM TrisHCl pH 8.0 |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | overnig |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Cell pellets from 100ml cultures were resuspended in 3ml of 20mM TrisHCl pH 8.0, 5mM EDTA, 1mM PMSF. The cells were sonicated (6x20sec) then centrifuged (8000g, 30min). The pellets were vortex to a homogenous suspension in wash buffer, then shaken at reoom temperature for 10min before being centrifuged (14000rpm, 5min). This wash step was then repeated with 4M urea in the buffer. Finally the pellets were dissolved in solubilization buffer and applied to a HPLC DEAE column. Proteins were eluted in 6M urea by a linear gradient of NaCl. The protein waxs refolded was achieved by sequential dialysis from 6M to 4M to 2M urea in refolding buffer. The protein (0.1-0.2mg/ml) was dialyzed against 6.7 volumes of 4M urea in wash buffer for 30min at room temperature, followed by one change of the dialysis solution and dialyzed for an additional 30min. This was then repeated with 2M urea in refolding buffer. The final dialysis was performed against refolding buffer with two initial changes of buffer for 30min and then finaly overnight at 4degC. |
| Refolding Assay | Far-UV Circular Dichroism,Amino acid sequencing,ELISA |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |