Refolding Record:
| Protein | |
|---|---|
| Protein Name | Daunorubicin resistance protein |
| Abbreviated Name | DrrC |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Streptomyces peucetius |
| UniProt Accession | Q54827 |
| 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 | 785 |
| Molecular Weight | 86038.6 |
| Pi | 6.56405 |
| Molecular Weight | 86038.6 |
| Disulphides | 0 |
| Full Sequence |
MGHHHHHHHHHHSSGHIEGRH MQQQAIQIIG ARENNLKGIS VEVPKHKVTV FTGVSGSGKS SLVFDTIAAE SQRQLNETFT AFVRNRLPKY GQPDVDDIAN LSAAVIIDQK RIGGNARSTV
GTITDIYALL RLLYSRVGKP WIGYSNAFSF NDPSGMCPDC EGLGHRVQVD LDKLLDRSKS LNEGAIRHPA FNVGGWFWKL YANSGLFDND KKLRDYTESE WQAFLYGAQG SIALEWQGGK VNSKYEGLMD KFNRLYLRKE PDEMSAKNRV ALQQVVTRGP CDTCKGQRLS QRALGCRING RNIAELASLE IADLMEAVKK
VDTSSVSTLT ASLIERLQHL IELGLGYLSL NRETSTLSGG ESQRIKMVRH LNSSLVDMVY IFDEPTIGLH PSDVQRLSRL LMELAEKGNT VLVVEHDRDI IEVAEHVIDI GPGAGRHGGE IVYQGDVRGL TEADTPTGRF LRRIIPIKER FRGPRGHLEV TGARIHNLKD ISVRIPLGVL TAVTGPAGSG KSTLIHDVLL
RQHPSAVVID QSAVTTNRRS NPATYTGIMD EIRRLFAREN QVSPSLFSFN SEGACPTCQG HGIIYTDLAF MDPMITTCEV CAGMRFTDDV LRLTLRGQNI HDVLSMSAAE AAEFFTEPKV AKTLRSINQV GLGYLQLGQP LNTLSGGECQ RIKLATELGK KGSVYVMDEP TTGLHMSDVD SLVHLVDGLV EKGDSVIVIE
HNLDVVKHAD WVVDLGPGGG SEGGRVIFEG TPADLAEAEH SVTGRFLAAS LAGAGSEADH RARR
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Furuya K and Hutchinson CR (1998) Gene, 168, 243-249 |
| Project Aim | Functional Studies |
| Fusion | N-terminal hexahistidine tag |
| 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 | 3h |
| Expression Vector | pET16b |
| Expression Protocol | no details provided |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 8M urea, 25mM TrisPO4 pH 7.5, 0.2M KCl, 1mM EDTA, 10mM DTT |
| Refolding Buffer | 50mM HEPES pH 7.5, 0.2M KCL, 100microM zinc acetate, 200microM DTT, 20% glycerol (w/v) |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 15h |
| Redox Agent | DTT |
| Redox Agent Concentration | 0.2mM,0.2mM |
| Refolding Protocol | After expression, cells were lysed by sonication and then centrifuged (10min, 10000g, 4degC). The supernatant was centrifuged again (1hr, 120000g, 15degC), and the pellet (60microg) was resuspended in 2ml solubilization buffer. The solubilized protein was dialyzed against 200ml 8M urea, 1mM Tris-acetate pH 7.0, 0.2M KCl, 1mM DTT for 4h at room temperature, then dialyzed 3 times against 200ml refolding buffer for 5h each 4deg. The dialysate was centrifuged (5min, 14000rpm, 4degC) and the supernatant stored at -80degC |
| Refolding Assay | DNA binding |
| Refolding Chaperones | None |
| Refolding Additives | None,Glycerol |
| Additives Concentration | 20% |
| Refolding Yield | |
| Purity | |
| Notes | See Furuya and Hutchinson (1996), J Bacteriology, 178:6310-6318 for more details of expression protocol (same as for DnrN protein) |