Refolding Record:
| Protein | |
|---|---|
| Protein Name | Duffy binding protein |
| Abbreviated Name | DBP |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Plasmodium Vivax |
| UniProt Accession | P22290 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | Receptor-binding domain aa.126-509 |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 397 |
| Molecular Weight | 46215.3 |
| Pi | 8.64 |
| Molecular Weight | 46215.3 |
| Disulphides | Unknown |
| Full Sequence |
GEEKDGEHKT DSKTDNGKGA DNLVMLDYET SSNGQPAGTL DNVLEFVTGH EGNSRKNSSN GGNPYDIDHK KTISSAIINH AFLQNTVMKN CNYKRKRRER DWDCNTKKDV CIPDRRYQLC MKELTNLVNN TDTNFHRDIT FRKLYLKRKL IYDAAVEGDL LLKLNNYRYN KDFCKDIRWS LGDFGDIIMG TDMEGIGYSK VVENNLRSIF GTDEKAQQRR KQWWNESKAQ IWTAMMYSVK KRLKGNFIWI CKINVAVNIE PQIYRWIREW GRDYVSELPT EVQKLKEKCD GKINYTDKKV CKVPPCQNAC KSYDQWITRK KNQWDVLSNK FISVKNAEKV QTAGIVTPYD ILKQELDEFN EVAFENEINK RDGAYIELCV CSV DKLAAALDHHHHHH
|
| Notes | Sequence inferred from original UniProt record, inconjunction with Q26145_PLAVI (Duffy receptor binding domain) and general ivormation provided in paper |
| Expression | |
|---|---|
| Report | Yazdani SS, Shakri AR, Chitnis CE (2004) biotechnology letters, 26, 1891-1895 |
| Project Aim | Recombinant Protein Expression |
| Fusion | C-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BLR(DE3)pLysS |
| Expression Temp | 37.0 |
| Expression Time | 4h |
| Expression Vector | pET28a+ |
| Expression Protocol | See paper for details of media composition and conditions. 10ml primary seed culture was used to inoculate two 200ml secondary seed cultures and grown a further 7-8h at 37degC. Both flasks of secondary seed cultures were then used to inoculate 10L of medium in a bioractor. Culture was induced with 1mM IPTG and cells were harvested after 4h incubation |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 6M GdnHCl |
| Refolding Buffer | 10mM TriHCl pH 7.2, 1mM EDTA, 0.5mM arginine, 1M urea, 2mM GSH, 0.2mM GSSG |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 7.2 |
| Refolding Temperature | 10.0 |
| Protein Concentration | 20microg/ml |
| Refolding Time | 36h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 2mM/0.2mM |
| Refolding Protocol | 30g of cell pellet was lysed by sonication and inclusion bodies were collected by centrifugation (12000g). Inclusion bodies werer solubilized in 6M GdnHCl and the protein was purified under denaturing conditions by metal affinity chromatography using Ni-NTA matix. The purified denatured protein was diluted 40-fold into 2L of pre-cooled refolding buffer to a final concentration of 20microg/ml and left for 36h without stirring at 10degC. After refolding, the solution was concentrated and the buffer exchanged with diafiltration buffer (50mM phosphate pH 6.5, 1M urea) and then loaded on 10ml of SP-sepharose fast flow matrix. The protein was eluted with 200mM NaCl. |
| Refolding Assay | Ligand Binding,SDS-PAGE,Reverse phase chromatography |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 0.5mM |
| Refolding Yield | n/a |
| Purity | 98% |
| Notes | n/a |