Refolding Record:
| Protein | |
|---|---|
| Protein Name | Serine-repeat antigen protein |
| Abbreviated Name | SERA |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Plasmodium falciparum |
| UniProt Accession | Q9TY95 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | SERA5 |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 438 |
| Molecular Weight | 50790.5 |
| Pi | 4.69 |
| Molecular Weight | 50790.5 |
| Disulphides | Unknown |
| Full Sequence |
TEDDDEDDYTEYKLTESIDNILVKMFKTNENNDKSELIKLEEVDDSLKLELMNYCSLLKDVDTTGTLDNYGMGNEMDIFNNLKRLLIYHSEENINTLKNKFRNAAVCLKNVDDWIVNKRGLVLPELNYDLEYFNEHLYNDKNSPEDKDNKGKGVVHVDTTLEKEDTLSYDNSDNMFCNKEYCNRLKDENNCISNLQVEDQGNCDTSWIFASKYHLETIRCMKGYEPTKISALYVANCYKGEHKDRCDEGSSPMEFLQIIEDYGFLPAESNYPYNYVKVGEQCPKVEDHWMNLWDNGKILHNKNEPNSLDGKGYTAYESERFHDNMDAFVKIIKTEVMNKGSVIAYIKAENVMGYEFSGKKVQNLCGDDTADHAVNIVGYGNYVNSEGEKKSYWIVRNSWGPYWGDEGYFKVDMYGPTHCHFNFIHSVVIFNVDLPMNN
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hodder AN, Drew DR, Epa VC, Delorenzi M, Bourgon R, Miller SK, Moritz RL, Frecklington DF, Simpson RJ, Speed TP, Pike RN, Crabb BS. (2003) Biologycal Chemistry, 278, 48169-48177 |
| Project Aim | Drug 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 | 0.0 |
| Expression Time | 00 |
| Expression Vector | pPROEXHTb |
| Expression Protocol | The enzyme domain of SERA5 was amplified from P. falciparum (D10) genomic DNA using the oligonucleotides: 5\'-GGCGCGGATCCACAGAAGATGATGATGAAGATGATTATACTG-3\' and 5\'-GGCCGCTCGAGCTAATTATTCATAGGTAAATCAACATTGAATATAAC-3\'. PCR products were digested with BamHI and XhoI (underlined above), ligated into pPROEXHTb (Invitrogen, Carlsbad, CA), and transformed into Escherichia coli strain BL21 cells (Stratagene, CA). |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 6 M guanidine-HCl/250 mM NaCl/20 mM Tris, pH 8.0, and 8 M urea/250 mM NaCl/20 mM Tris, pH 8.0 |
| Solubilization Buffer | 6 M guanidine-HCl/250 mM NaCl/10 mM imidazole/20 mM Tris, pH 8.0, buffer containing 20 mM -mercaptoethanol |
| Refolding Buffer | 2 M urea/100 mM NaCl/20 mM Tris, pH 8.0, containing 1 mM reduced glutathione and 0.25 mM oxidized glutathione |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 0.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 1/0.25 mM |
| Refolding Protocol | Washed inclusion bodies were solubilized in 6 M guanidine-HCl/250 mM NaCl/10 mM imidazole/20 mM Tris, pH 8.0, buffer containing 20 mM -mercaptoethanol. The solution was clarified by filtration through a 1-µm glass filter (Pall-Gelman, Ann Arbor, MI) then incubated in batch mode overnight with nickel-nitrilotriacetic acid-agarose (Ni-NTA) resin (Qiagen, GmbH, Hilden, Germany). After washes (10 column volumes each) with 6 M guanidine-HCl/250 mM NaCl/20 mM Tris, pH 8.0, and 8 M urea/250 mM NaCl/20 mM Tris, pH 8.0, the bound 5PE was eluted with 8 M urea/1 M imidazole/20 mM Tris, pH 8.0, containing 20 mM -mercaptoethanol. The Ni-NTA-purified 5PE was refolded by dilution into 2 M urea/100 mM NaCl/20 mM Tris, pH 8.0, containing 1 mM reduced glutathione and 0.25 mM oxidized glutathione. The refolded protein was concentrated and purified by anion-exchange chromatography. Bound protein was eluted from a 1-ml Hitrap Q strong anion-exchange column (Amersham Biosciences, Uppsala, Sweden) using a linear gradient from 1% to 100% buffer B over 30 min at a flow rate of 2 ml/min. Buffer A comprised 20 mM Bis-Tris, pH 6.50, and buffer B comprised 1 M NaCl and 20 mM Bis-Tris, pH 6.50. All solvents were 0.22-µm-filtered prior to use. Gel permeation chromatography was used as a final polishing step to remove traces of aggregate. A Hiload 16/60 Superdex 200 preparative column (Amersham Biosciences) was used throughout. Gel permeation chromatography was performed using 500 mM NaCl/50 mM Na2HPO4, pH 7.50 buffer. Gel filtration standards (Bio-Rad Laboratories, Richmond, CA) were used to construct a calibration curve for the subsequent determination of the relative molecular weight of sera 5PE. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |