Refolding Record:
| Protein | |
|---|---|
| Protein Name | Apical membrane antigen 1 |
| Abbreviated Name | AMA1 |
| SCOP Family | Apical membrane antigen 1 |
| Structure Notes | |
| Organism | Plasmodium falciparum |
| UniProt Accession | P50492 |
| 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 | 598 |
| Molecular Weight | 69135.1 |
| Pi | 5.52 |
| Molecular Weight | 69135.1 |
| Disulphides | 8 |
| Full Sequence |
QNYWEHPYQKSDVYHPINEHREHSKEYEYPLHQEHTYQQEDSGEDENTLQHAYPIDHEGAEPAPQEQNLFSSIEIVERSNYMGNPWTEYMAKYDIKEVHGSGIRVDLGEDAEVAGTQYRLPSGKCPVFGKGIIIENSNTTFLKPVATGNQDLKDGGFAFPPTNPLISPMTLDHMRDFYKNNEYVKNLDELTLCSRHAGNMNPDNDKNSNYKYPAVYDYNDKKCHILYIAAQENNGPRYCNKDESKRNSMFCFRPAKDKSFQNYTYLSKNVVDNWEKVCPRKNLENAKFGLWVDGNCEDIPHVNEFSANDLFECNKLVFELSASDQPKQYEQHLTDYEKIKEGFKNKNASMIKSAFLPTGAFKADRYKSRGKGYNWGNYNRKTQKCEIFNVKPTCLINNSSYIATTALSHPNEVEHNFPCSLYKDEIKKEIERESKRIKLNDNDDEGNKKIIAPRIFISDDIDSLKCPCDPEIVSNSTCNFFVCKCVEKRAEVTSNNEVVVKEEYKDEYADIPEHKPTYDKMKIIIASSAAVAVLATILMVYLYKRKGNAEKYDKMDEPQDYGKSNSRNDEMLDPEASFWGEEKRASHTTPVLMEKPYY
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Gupta A, Bai T, Murphy V, Strike P, Anders RF, Batchelor AH. (2005) Protein Expression and Purification, 41, 186-198 |
| Project Aim | Protein refolding |
| 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 | 37.0 |
| Expression Time | 3 h |
| Expression Vector | pBB117-pBB119 |
| Expression Protocol | Protein expression Expression plasmids pBB117 and pBB119 were transformed into Escherichia coli BL21(DE3) cells containing the supplementary rare tRNA expression plasmid pRIL (Stratagene) and selected on Luria broth-agar plates containing 100 μg/ml ampicillin (to select for pBB117 and pBB119) and 30 μg/ml tetracycline to select for pRIL. Log phase cells were frozen in 10% glycerol to act as starter cultures. Ten millilitres of Luria broth (LB, 5 g yeast extract, 10 g tryptone, 10 g NaCl/L) supplemented with ampicillin and tetracycline was innoculated with 5 μl of frozen stock culture and grown to confluence overnight. Ten millilitre overnight cultures were added to 1 L of LB containing ampicillin and tetracycline and incubated with shaking at 37 °C for 3 h. Cultures were then induced with 1 mM IPTG for a further 3 h. Some of the protein preparations were made utilizing richer media (35 g tryptone, 20 g yeast extract, 5 g NaCl/L) or media supplemented with 2% glucose. However, use of richer media made little difference to protein yields (Fig. 3). Moreover, the pRIL plasmid was not necessary for AMA1 expression but was used for most protein preparations because it improved protein yields by about 25% (data not shown). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | Tris–HCl, pH 8, 0.5 mM EDTA, 2 M urea |
| Solubilization Buffer | 200 ml of 20 mM Tris–HCl, pH 8, 2 mM of 2-mercaptoethanol (2ME), and 6 M guanidine hydrochloride (GdnHCl) |
| Refolding Buffer | 20 mM Tris–HCl, pH 8, 6 M GdnHCl |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a |
| Refolding Protocol | Protein solubilization As expected proteins resulting from pBB117, pBB119, and pBB120, expressed to high yields (approximately 0.2 mg/ml LB), but were insoluble. One litre LB cell pellets were stored frozen and resuspended in 200 ml of 30 mM Tris–HCl, pH 8, 200 mM NaCl, 2 mM EDTA, 0.2 mM PMSF, and 0.1% Triton X-100, by pipetting, treatment with 0.1 mg lysozyme and sonication. Most protein impurities were solubilized by this wash buffer. AMA1 proteins remained insoluble and were repelleted by centrifugation at 8000g for 30 min. The AMA1 protein pellets were then washed a second time by sonination into 20 mM Tris–HCl, pH 8, 0.5 mM EDTA, 2 M urea and repelleted. The importance of this second wash step is uncertain. We included it because we were concerned about carry over of Triton X-100 into the refolding process. Examination of the AMA1 proteins by reduced SDS–PAGE indicated bands of approximately 80% purity (Fig. 3). However, if samples were not reduced the band was smeared and largely consisted of higher molecular weight disulphide-linked multimers of misfolded AMA1 (Fig. 4). The proteins were resolubilized in 200 ml of 20 mM Tris–HCl, pH 8, 2 mM of 2-mercaptoethanol (2ME), and 6 M guanidine hydrochloride (GdnHCl) by rotating the centrifugation bottles at room temperature overnight. In some preparations 2ME was not used at this stage. In the absence of 2ME approximately 50% of the AMA1 pellet was not solubilized in GdnHCl. The use of reduced or non-reduced protein dissolved in GdnHCl made no difference to subsequent refolding. Protein refolding AMA1 proteins were diluted into 20 mM Tris–HCl, pH 8, 6 M GdnHCl such that the final protein concentration was 20 μg/ml. Fifty millilitres of dilute protein was dialysed against 3.5 L of 20 mM Tris–HCl, pH 8, 0.5 mM EDTA, 1 M urea, 200 mM NaCl, 2 mM 2ME, and 0.2 mM cystamine–HCl overnight at 4 °C. The dialysis bag was then transferred into 3.5 L of 20 mM acetic acid–NaOH, pH 5, 0.5 mM EDTA for a further overnight dialysis. At this stage a significant proportion of the AMA1 (approximately 20%) had precipitated out of solution, but this consisted of multimeric misfolded AMA1. Sodium azide (0.02%) was added to dialysed samples that were stored at 4 °C for up to 2 months. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |