Refolding Record:
| Protein | |
|---|---|
| Protein Name | Merozoite Surface Protein-1 |
| Abbreviated Name | MSP-1 |
| SCOP Family | Merozoite surface protein 1 (MSP-1) |
| Structure Notes | |
| Organism | Plasmodium Vivax |
| UniProt Accession | Q2I7F0 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 1745 |
| Molecular Weight | 195935.0 |
| Pi | 6.51 |
| Molecular Weight | 195935.0 |
| Disulphides | Unknown |
| Full Sequence |
MKALLFLFSFIFFVTKCQCETESYKQLVAKLDKLEALVVDGYELFHKKKLGESDIQVETNASANNNNNNQVSVLTSKIRNFVGKFLELQIPGHTDLLHLIRELAFEPNGIKYLVESYEEFNQLMHVINFHYDLLRAKLHDMCAHDYCKIPEHLKITEKELDMLKKVVLGYRKPLDNIKDDIGKLEAFITKNKNTIKKISDLITEEHKKRSGQYTSTISGNGAQTDGHQPTTASSETSSGSSVSSVSGSSGLGSSGTGSTGTGSTGNISPSQARADSPSTGTDYNAKKIIFQAVYNTIFYTNQLQEAQKLIAVLEKRVKVLKEHKDIKVLLEQVAKEKEKLPKDNTTTTNLTNVHKEAESKIAELEKKIEAIAKTVNFDLDGLFTDAEELEYYLREKAKMAGTLIIPESTKSAGTPGKTVPTLKETYPHGISYALAENSIYELIEKIGSDETFGDLQNPDDGKQPKKGILINETKRKELLEKIMNKIKIEEDKLPNLKKEYDEKFKVYEEKVKDFQPAFNHFYEARLDNTLVENKFDEFKTKREAYMEEKKKLESCSYEQNTNLINKLKKQLTYLEDYVLRKDIADDEIKHFSFMEWKLKSEIYDLAQEIRKNENKLTVENKFDFSGVVELQVQKVLIIKKIEALKNVQNLLKNAKVKDDLYVPKVYKTGEKPEPYYLMVLKREIDKLKDFIPKIESMIATEKNKPTVAAADIVAKGQSLRGASETGTTGNTVNAQTAVVQPPQYQVVNAVTVQPGTTGHQAQGGEAETQTNSVQAAQVQQTPAGAGGQVASTQTTSQAPAPTQASPEPAPAAPPSTPAAAVAPAPTMSKLEYLQKLLDFLKSAYACHKHIFVTNSTMDKKLLKEYELNADEQNKIKENKCDELDLLFNVQNNLPAMYSIYDSMSNELQNLYIELYQKEMVYNIYKNKDTDKKIKAFLETSNNKAAAPAQSAAKPSGQAGTTPVTTTVPVTTTTVTPSPQTSVVTSTPPTPQAEENQRVGGNSEEKPEADTAQVEKFYEKHSQIDKYNDYFKKFLESKKEEIIKMDDTKWNALGKEIEELKKKLQVSLDHYGKYKLKLERFLKKKNKISNSKDQIKKLTSLKNKLERRQNLLNNPTSVLKNYTAFFNKKRETEKKEVENTLKNTEILLKYYKARAKYYIGEPFPLKTLSEESMQKEDNYLNLEKFRVLSRLEGRLGKNIELEKENISYLSSGLLHVLTELKEIINDKKYSGKDHAKNIAEVKKALQAYQELIPKVTSQESTSVAVTVPGAVVPGVPTAAAAGSGASGAVPPAGGPSPPATGGVVPGVVESAEAQTQTQAQDYAEDYDKVIALPLFGNNDDDGEEDQVTTGEAESEAPEILVPAGISDYDVVYLKPLAGMYKTIKKQLENHVNAFNTNITDMLDSRLKKRNYFLEVLNSDLNPFKYSSSGEYIIKDPYKLLDLEKKKKLIGSYKYIGASIDMDLATANDGVAYYNKMEELYKTHLTAVNAQIKKVEDDINTQNEELKKIENEANKTAEKAKFTAKKAELEKYLPFLNSLQKEYESLVSKVNTYTDNLKKVINNCQLEKKEAEITVKKLQDYNKMDEKLEEYKKSEKKNEVKSSGLLEKLMKSKLIKENESKEILSQLLNVQTQLLTMSSEHTCIDTNVPDNAACYRYLDGTEEWRCLLTFKEEGGKCVPASNVTCKDNNGGCAPEAECKMTDSNKIVCKCTKEGSEPLFEGVFCSSSSFLSLSFLLLMLLFLLCMEL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Dutta S, Kaushal DC, Ware LA, Puri SK, Kaushal NA, Narula A, Upadhyaya DS, Lanar DE. (2005) Infection and Immunity, 73, 5936-5944 |
| Project Aim | Vaccine 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 | 25.0 |
| Expression Time | 2 h |
| Expression Vector | pETAT |
| Expression Protocol | Expression and purification of refolded PcMSP-142 and PvMSP-142 proteins. The recombinant plasmids carrying the PcMSP-142 or PvMSP-142 gene were transformed into E. coli BL-21(DE3) strain cells (Novagen, San Diego, CA). The fermentation and induction conditions were identical to those described above for soluble protein expression. To allow upscaling of the procedure and evaluation of different lots of material, the amount of all buffers and resins were indexed to a starting cell weight (scw). Cell paste from both PcMSP-142 and PvMSP-142 fermentations were suspended (10 ml g–1 scw) in resuspension buffer (RB; 15 mM sodium phosphate, 450 mM NaCl, pH 7.4). Cells were lysed by microfluidization, and the insoluble inclusion bodies were harvested by centrifugation (12,000 x g, 45 min, 4°C). The pellet was suspended in cold RB (10 ml g–1 scw) using a manual glass tissue homogenizer, and the suspension was centrifuged as before |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Microfluidizer |
| Lytic Agent | None |
| Pre-Refolding Purification | Ni-NTA agrose chromatography |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | RB; 15 mM sodium phosphate, 450 mM NaCl, pH 7.4 |
| Solubilization Buffer | SB; 7 M urea in RB |
| Refolding Buffer | 20 mM sodium phosphate, 1 mM reduced glutathione [GSH], 0.05 mM oxidized glutathione [GSSG]; pH 8.0 |
| Pre-Refolding Purification | Ni-NTA agrose chromatography |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 22.0 |
| Protein Concentration | n/a |
| Refolding Time | 16 h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 1/0.05 mM |
| Refolding Protocol | The washed pellet was resuspended in solubilization buffer (SB; 7 M urea in RB) (6.6 ml g–1 scw). The pellet was suspended by homogenization, incubated for 20 min on a shaking platform at room temperature (RT), and centrifuged (12,000 x g, 45 min, 10°C). A nickel-nitrilotriacetic acid (Ni-NTA) agarose (QIAGEN, Valencia, CA) column (0.6 ml gm–1 scw) was equilibrated with SB, and the cleared supernatant was loaded onto the column. The column was washed with 10 column volumes (CV) of SB containing 20 mM imidazole (pH 7.4), and protein was eluted with 5 CV of SB containing 500 mM imidazole (pH 7.4). The Ni-NTA agarose column-eluted protein was refolded by a rapid 20-fold dilution in refolding buffer (20 mM sodium phosphate, 1 mM reduced glutathione [GSH], 0.05 mM oxidized glutathione [GSSG]; pH 8.0). After refolding (16 h, 22°C) in sealed bottles, the protein was bound to a Q-Sepharose resin column (0.6 ml gm–1 scw) pre-equilibrated with 20 mM sodium phosphate (pH 8.0). The Q-Sepharose resin was washed with 10 CV of 20 mM phosphate buffer (pH 8.0), followed by 5 CV of the same buffer containing 50 mM NaCl (pH 8.0). The PvMSP-142 or PcMSP-142 protein was eluted from the column in 20 mM sodium phosphate and 200 mM NaCl (pH 8.0). The fractions containing the protein were pooled and dialyzed against PBS. The endotoxin content of the final product was estimated by a chromogenic Limulus amebocyte lysate (LAL) endpoint assay (Associates of Cape Cod, Falmouth, MA) following manufacturer\'s instructions. |
| Refolding Assay | Western Blot |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |