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 | P04934 |
| 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 | 1688 |
| Molecular Weight | 191500.0 |
| Pi | 5.96 |
| Molecular Weight | 191500.0 |
| Disulphides | Unknown |
| Full Sequence |
VTHESYQELVKKLEALEDAVLTGYGLFHKEKMILNEEEITTKGASAQSGTSGTSGTSGTSGTSGTSGTSAQSGTSGTSAQSGTSGTSAQSGTSGTSGTSGTSPSSRSNTLPRSNTSSGASPPADASDSDAKSYADLKHRVRNYLFTIKELKYPELFDLTNHMLTLCDNIHGFKYLIDGYEEINELLYKLNFYFDLLRAKLNDVCANDYCQIPFNLKIRANELDVLKKLVFGYRKPLDNIKDNVGKMEDYIKKNKTTIANINELIEGSKKTIDQNKNADNEEGKKKLYQAQYDLSIYNKQLEEAHNLISVLEKRIDTLKKNENIKELLDKINEIKNPPPANSGNTPNTLLDKNKKIEEHEEKIKEIAKTIKFNIDSLFTDPLELEYYLREKNKKVDVTPKSQDPTKSVQIPKVPYPNGIVYPLPLTDIHNSLAADNDKNSYGDLMNPDTKEKINEKIITDNKERKIFINNIKKQIDLEEKKINHTKEQNKKLLEDYEKSKKDYEELLEKFYEMKFNNNFDKDVVDKIFSARYTYNVEKQRYNNKFSSSNNSVYNVQKLKKALSYLEDYSLRKGISEKDFNHYYTLKTGLEADIKKLTEEIKSSENKILEKNFKGLTHSANASLEVYDIVKLQVQKVLLIKKIEDLRKIELFLKNAQLKDSIHVPNIYKPQNKPEPYYLIVLKKEVDKLKEFIPKVKDMLKKEQAVLSSITQPLVAASETTEDGGHSTHTLSQSGETEVTEETEETEETVGHTTTVTITLPPKEVKVVENSIEHKSNDNSQALTKTVYLKKLDEFLTKSYICHKYILVSNSSMDQKLLEVYNLTPEEENELKSCDPLDLLFNIQNNIPAMYSLYDSMNNDLQHLFFELYQKEMIYYLHKLKEENHIKKLLEEQKQITGTSSTSSPGNTTVNTAQSATHSNSQNQQSNASSTNTQNGVAVSSGPAVVEESHDPLTVLSISNDLKGIVSLLNLGNKTKVPNPLTISTTEMEKFYENILKNNDTYFNDDIKQFVKSNSKVITGLT
ETQKNALNDEIKKLKDTLQLSFDLYNKYKLKLDRLFNKKKELGQDKMQIKKLTLLKEQLESKLNSLNNPHNVLQNFSVFFNKKKEAEIAETENTLENTKILLKHYKGLVKYYNGESSPLKTLSEVSIQTEDNYANLEKFRVLSKIDGKLNDNLHLGKKKLSFLSSGLHHLITELKEVIKNKNYTGNSPSENNKKVNEALKSYENFLPEAKVTTVVTPPQPDVTPSPLSVRVSGSSGSTKEETQIPTSGSLLTELQQVVQLQNYDEEDDSLVVLPIFGESEDNDEYLDQVVTGEAISVTMDNILSGFENEYDVIYLKPLAGVYRSLKKQIEKNIFTFNLNLNDILNSRLKKRKYFLDVLESDLMQFKHISSNEYIIEDSFKLLNSEQKNTLLKSYKYIKESVENDIKFAQEGISYYEKVLAKYKDDLESIKKVIKEEKEKFPSSPPTTPPSPAKTDEQKKESKFLPFLTNIETLYNNLVNKIDDYLINLKAKINDCNVEKDEAHVKITKLSDLKAIDDKIDLFKNHNDFEAIKKLINDDTKKDMLGKLLSTGLVQNFPNTIISKLIEGKFQDMLNISQHQCVKKQCPENSGCFRHLDEREECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGKKITCECTKPDSYPLFDGIFCS
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Leung WH, Meng ZQ, Hui G, Ho WK. (2004) Biochemica et Biophysica Acta, 1675, 62-70 |
| Project Aim | Vaccine studies |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | origami B(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 3 h |
| Expression Vector | pET-32a |
| Expression Protocol | A single transformed Origami (DE3) colony was grown overnight in 10-ml LBA medium at 378C with shaking at 250 rpm. The overnight culture was inoculated into 500-ml LBA medium and the culture was allowed to grow for about 3 h at 378C with shaking at 250 rpm to achieve an O.D.600 of 0.6–0.8. At this point, IPTG was added to a final concentration of 0.1 mM to induce protein expression. After 3 h, the culture was harvested by centrifugation. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6-0.8 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50 mM Tris–HCl (pH 8.0) containing 1 mM EDTA and 1% (v/v) Triton-X 100; afterwards, a final wash with (20 ml) 50 mM Tris–HCl (pH 8.0) containing 1 mM EDTA and 0.5 M Guanidine HCl |
| Solubilization Buffer | 20 ml of 100 mM Tris–HCl (pH 12.5) containing 2 M urea |
| Refolding Buffer | 80 ml dH2O (fivefold dilutions). The pH of the diluted sample was adjusted to 8.0 with 5 M HCl |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The bacterial cell pellet was first resuspended in a sonication buffer (20 ml) and then sonicated by cycles of 30-s sonication followed by cooling until the solution became translucent. After centrifugation at 13 000 rpm for 20 min, the pellet was washed (20 ml) three times with 50 mM Tris–HCl (pH 8.0) containing 1 mM EDTA and 1% (v/v) Triton-X 100; afterwards, a final wash with (20 ml) 50 mM Tris–HCl (pH 8.0) containing 1 mM EDTA and 0.5 M Guanidine HCl. The inclusion bodies obtained were solubilized and renatured using the method described by Patra et al. [31]. Briefly, 20 ml of 100 mM Tris–HCl (pH 12.5) containing 2 M urea was used to solubilize the inclusion bodies at 48C with vigorous shaking overnight. The sample was centrifuged at 13 000 rpm for 20 min at 48C. The supernatant was collected and then diluted immediately with 80 ml dH2O (fivefold dilutions). The pH of the diluted sample was adjusted to 8.0 with 5 M HCl. The sample was then dialyzed extensively in 20 mM Tris–HCl (pH 8.0) to remove the remaining urea. The sample was filtered by a 0.45-Am filter and then passed through a Ni2+ ion charged chelating Sepharose fast flow (10 ml gel matrix) column (Amersham Biosciences). The column was washed with three bed volumes of 20 mM Tris–HCl (pH 8.0) containing 40 mM imidazole. |
| Refolding Assay | ELISA |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 48 mg |
| Purity | n/a |
| Notes | n/a |