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 falciparum |
| UniProt Accession | P04933 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Small Proteins |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 372 |
| Molecular Weight | 42734.7 |
| Pi | 5.73 |
| Molecular Weight | 42734.7 |
| Disulphides | Unknown |
| Full Sequence |
AISVTMDHILSGFENEYDVIYLKPLAGVYRSLKKQIEKNIFTFNLNLNDILNSRLKKRKYFLDVLESDLMQFKHISSNEYIIEDSFKLLNSEQKNTLLKSYKYIKESVENDIKFAQEGISYYEKVLAKYKDDLESIKKVIKEEKEFPSSPPTTPPSPAKTDEQKKESKFLPFLTNIETLYNNLVNKIDDYLINLKAKINDCNVEKNEAHVKITKLSDLKAIDDKIDLFKNHNDFEAIKKLINDDTKKDMLGKLLSTGLVQNFPNTIISKLIEGKFQDMLNISQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Epp C, Kauth CW, Bujard H, Lutz R. (2003) J Chromatography B, 786, 61-72 |
| 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 | W3110Z1 |
| Expression Temp | 37.0 |
| Expression Time | 3 h |
| Expression Vector | pZE13/f-HX42 |
| Expression Protocol | For expression of msp-1D42 and msp-1F42 E. coli strain W3110Z1 [16] was transformed with plasmids pZE13/d-HX42 and pZE13/f-HX42, respectively. Overnight cultures derived from individual E. coli clones containing the proper expression vectors were diluted 1:50 in 1.5 l Luria-broth (LB) medium and grown with orbital shaking (220 rpm) at 37 °C in the presence of ampicillin (100 μg−1 ml−1) to log phase (OD600≈1). After induction with 1 mM IPTG for 3 h at 37 °C the cells were harvested by centrifugation for 10 min at 5000 g. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 1 = 600 |
| Cell Disruption Method | French Press |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 6 M GdmCl, 50 mM DTT, 50 mM Tris, pH 8.0 |
| Refolding Buffer | 1 M Arginin, 50 mM Tris, pH 8.0, 1 mM EDTA, 1 mM oxidized glutathione (GSSG), 10 mM reduced glutathione (GSH) |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 1 mg/ ml |
| Refolding Time | 12-16 h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 10/1 mM |
| Refolding Protocol | After resuspension of sedimented bacteria in PBS containing 20 u ml−1 DNaseI, 10 μg ml−1 Lysozyme, the cells were disrupted by French Press lysis at 1000 p.s.i.. The insoluble fraction containing more than 90% of the heterologously produced MSP-1 was seperated from the soluble material by centrifugation of the bacterial lysate at 40.000 g for 40 min and redissolved in 6 M GdmCl, 50 mM DTT, 50 mM Tris, pH 8.0 (4 ml g−1 pellet) by using an Ultraturrax homogenizer. The suspension was further incubated under stirring at RT for at least 2 h before non-solubilized material was sedimented (40.000 g for 40 min) and the supernatant dialysed against at least 100 volumes of 6 M GdmCl, 50 mM Tris, 2 mM EDTA, pH 3.0 at 4 °C. The final protein concentration was typically in the range of 25–30 mg ml−1. Protein renaturation was performed at 4 °C according to a procedure described by Rudolph and Fischer [17] commonly referred to as “pulse renaturation”. In short, the solubilized protein at a concentration of 10 mg ml−1 was repeatedly diluted 1:100 in refolding buffer (1 M Arginin, 50 mM Tris, pH 8.0, 1 mM EDTA, 1 mM oxidized glutathione (GSSG), 10 mM reduced glutathione (GSH)) in 1.5 h intervals until a final protein concentration of 1 mg ml−1 was reached. After the final “pulse” the mixture was kept at 4 °C for 12–16 h before it was dialysed twice against 50 volumes of 50 mM Tris, pH8.0, 300 mM NaCl, 10% glycerol, 10 mM imidazole. |
| Refolding Assay | ELISA |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 1 M |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |