Refolding Record:
| Protein | |
|---|---|
| Protein Name | Plasmepsin 2 |
| Abbreviated Name | n/a |
| SCOP Family | Pepsin Like |
| Structure Notes | |
| Organism | Plasmodium falciparum |
| UniProt Accession | Q8I6V3 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 453 |
| Molecular Weight | 51480.8 |
| Pi | 5.35 |
| Molecular Weight | 51480.8 |
| Disulphides | Unknown |
| Full Sequence |
MDITVREHDFKHGFIKSNSTFDGLNIDNSKNKKKIQKGFQILYVLLFCSVMCGLFYYVYENVWLQRDNEMNEILKNSEHLTIGFKVENAHDRILKTIKTHKLKNYIKESVNFLNSGLTKTNYLGSSNDNIELVDFQNIMFYGDAEVGDNQQPFTFILDTGSANLWVPSVKCTTAGCLTKHLYDSSKSRTYEKDGTKVEMNYVSGTVSGFFSKDLVTVGNLSLPYKFIEVIDTNGFEPTYTASTFDGILGLGWKDLSIGSVDPIVVELKNQNKIENALFTFYLPVHDKHTGFLTIGGIEERFYEGPLTYEKLNHDLYWQITLDAHVGNIMLEKANCIVDSGTSAITVPTDFLNKMLQNLDVIKVPFLPFYVTLCNNSKLPTFEFTSENGKYTLEPEYYLQHIEDVGPGLCMLNIIGLDFPVPTFILGDPFMRKYFTVFDYDNQSVGIALAKKNL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hill J, Tyas L, Phylip LH, Kay J, Dunn BM, Berry C. (1994) FEBS Letters, 352, 155-158 |
| Project Aim | Expression and charactrization |
| Fusion | N-terminal T7 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 | 2 h |
| Expression Vector | pET3a |
| Expression Protocol | E. coli BLZl(DE3)pLysS cells transformed with the recombinant plasmid were grown to an Am of 0.4 in LB medium supplemented with 150 &/ml ampicillin, and were induced by the addition of isopropyl-B-D-thiogalactopyranoside (IPTG) to a final concentration of 0.4 mM.Incubation was continued at 37“C for a further 2 h, at which time the cells were harvested by centrifugation (3,000 x g for 10 min) and resuspended in TN buffer (50 mM Tris-HCl, pH 7.2, 0.15 M NaCl). Lysozyme was added (final concentration = 10 @ml) and the cells were lysed by freezing/thawing. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.4 = 600 |
| Cell Disruption Method | Freeze-thaw |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | relationships for both enzymes would facilitate the develop- |
| Solubilization Buffer | 6 M urea. 0.1 M Tris-HCI. DH 8.0. 1 mM glycine. 1 mM EDTA, 50 mM B-mercaptoethanol |
| Refolding Buffer | 10 mM Tris-HCl, pH 8.5 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 24 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Purification and refolding Lysed cells (from 1 1 of culture) were diluted to 200 ml in TN buffer and stirred overnight at 4°C. Insoluble material containing the recombinant protein was pelleted by centrifugation at 16,000 x ,g for 30 min. The pellet was resuspended in 200 ml buffer B (0.1 M Tris-HCl, pH 11.O, 50 mM j?-mercaptoethanol) and stirred at 4°C for 4 h. Insoluble material was re-pelleted and washed with a further 200 ml buffer B. After centrifugation, the washed pellet was resuspended in 10 ml buffer C (6 M urea. 0.1 M Tris-HCI. DH 8.0. 1 mM elvcine. 1 mM EDTA, 50 mM B-mercaptoethanol) add stirred ovemKht at’4”C to solubilise the recombinant material. Residual insoluble material was removed by centrifugation at 28,0000x g for 2 h. The supematant containing recombinant protein was rapidly diluted into 2 1 buffer D (10 mM Tris-HCl, pH 8.5) and stirred at 25°C for a further 24 h to allow refolding of the recombinant protein. This solution was concentrated to a volume of 30 ml using a Filtron Ultrasette, 5 kDa cut-off tangential flow concentrator (Flowgen Instruments Ltd, Sittingboume, Kent, UK) and the concentrate was loaded onto DEAE-cellulose Productiv PSClO-DE column (BPS Separations Ltd., Spennymoor, Co. Durham, UK) equilibrated in 0.1 M Tris-HCl buffer, pH 8.5. After extensive washing, the recombinant protein was eluted by a linear gradient (50 ml each) of O-0.8 M NaCl in the same buffer. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 20 mg/l culture |
| Purity | n/a |
| Notes | n/a |