Refolding Record:
| Protein | |
|---|---|
| Protein Name | Protective antigen |
| Abbreviated Name | PA |
| SCOP Family | Anthrax protective antigen |
| Structure Notes | |
| Organism | Bacillus anthracis |
| UniProt Accession | Q52NH4 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Membrane |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 764 |
| Molecular Weight | 85811.1 |
| Pi | 5.87 |
| Molecular Weight | 85811.1 |
| Disulphides | Unknown |
| Full Sequence |
MKKRKVLIPLMALSTILVSSTGNLEVIQAEVKQENRLLNESESSSQGLLGYYFSDLNFQAPMVVTSSTTGDLSIPSSELENIPSENQYFQSAIWSGFIKVKKSDEYTFATSADNHVTMWVDDQEVINKASNSNKIRLEKGRLYQIKIQYQRENPTEKGLDFKLYWTDSQNKKEVISSDNLQLPELKQKSSNSRKKRSTSAGPTVPDRDNDGIPDSLEVEGYTVDVKNKRTFLSPWISNIHEKKGLTKYKSSPEKWSTASDPYSDFEKVTGRIDKNVSPEARHPLVAAYPIVHVDMENIILSKNEDQSTQNTDSQTRTISKNTSTSRTHTSEVHGNAEVHASFFDIGGSVSAGFSNSNSSTVAIDHSLSLAGERTWAETMGLNTADTARLNANIRYVNTGTAPIYNVLPTTSLVLGKNQTLATIKAKENQLSQILAPNNYYPSKNLAPIALNAQDDFSSTPITMNYNQFLELEKTKQLRLDTDQVYGNIATYNFENGRVRVDTGSNWSEVLPQIQETTARIIFNGKDLNLVERRIAAVNPSDPLETTKPDMTLKEALKIAFGFNEPNGNLQYQGKDITEFDFNFDQQTSQNIKNQLAELNATNIYTVLDKIKLNAKMNILIRDKRFHYDRNNIAVGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYAVTKENTIINPSENGDTSTNGIKKILIFSKKGYEIG
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Gupta P, Waheed SM, Bhatnagar R. (1999) Protein Expression and Purification, 16, 369-76 |
| Project Aim | Over expression & Renaturation |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | SG13009[pRPEP4] |
| Expression Temp | 37.0 |
| Expression Time | 5 h |
| Expression Vector | pMW! |
| Expression Protocol | Expression of Protective Antigen For localizing the expressed recombinant protein in the cell, the E. coli strain SG13009 (pREP4) carrying the recombinant plasmid pMW1 was grown at 37°C in Luria broth with 100 􏰉g of ampicillin per milliliter and 25 􏰉g of kanamycin per milliliter at 250 rpm in 500ml flasks. When A600 reached 1.0, IPTG was added to a final concentration of 1 mM. After 5 h of induction, cells were harvested by centrifugation at 4000g for 20 min. Periplasm, cytosol, and inclusion bodies were checked for the presence of PA. To check for periplasmic localization the pellet from 100 ml culture was resuspended in 10 ml 30 mM Tris–Cl, EDTA 1 mM, 20 % sucrose, pH 8.0, and incubated in ice for 10 –15 min in 50-ml tubes. Sample was centrifuged at 8000g at 4°C for 10 min. Supernatant was removed and the pellet was resus- pended gently in 10 ml ice-cold 5 mM MgSO4 and incubated in ice for 10 min. Tubes were centrifuged at 8000g at 4°C for 10 min. The supernatant (periplasmic extract) was collected. To check for cytosolic localiza- tion of the recombinant protein, the pellet from 100 ml culture was resuspended in 5 ml of sonication buffer (50 mM Na-phosphate, pH 7.8, 300 mM NaCl). Cells were sonicated at 4°C (1-min bursts/1-min cooling/ 200 –300 W) for 5 cycles. The lysate was centrifuged at 10,000g for 30 min. Supernatant (cytosolic extract) was collected while the pellet (insoluble matter) was solubilized in 5 ml of 8 M urea, 0.1 M Na-phosphate, 0.01 M Tris–Cl, pH 8.0, by stirring for 1 h at room temperature. Tubes were centrifuged at 10,000g for 30 min, and the supernatant (inclusion bodies) was collected. Expression of PA was established by SDS–PAGE and Western blot analyses of the samples collected. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 1.0 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Ni-NTA column |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Nickel-chelating chromatography |
| Wash Buffer | n/a |
| Solubilization Buffer | 5 ml of 8 M urea, 0.1 M Na-phosphate, 0.01 M Tris–Cl, pH 8.0 |
| Refolding Buffer | 6 M GuHCl, 0.1 M Naphosphate, pH 7.8, and 300 mM NaCl |
| Pre-Refolding Purification | Ni-NTA column |
| Tag Cleaved | no tag |
| Refolding pH | 7.8 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 45 m |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | As PA was mainly localized in the inclusion bodies, the protein was purified under denaturing conditions. The pellet from 2 liters of culture was resuspended in 50 ml of buffer containing 6 M GuHCl, 0.1 M Na-phosphate, pH 7.8, and 300 mM NaCl. Cells were stirred at room temperature for 1 h. Lysate was centrifuged at 10,000g for 30 min at 4°C. The supernatant was mixed with 8 ml of 50% slurry of Ni-NTA resin and allowed to stir at room temperature for 45 min, and then the resin was loaded carefully into a 1.6-cm diam- eter column. The column was washed with 10 column vol of buffer containing 8 M urea, 0.1 M Na-phosphate, pH 7.8, and 300 mM NaCl. The resin was then washed with a gradient of 8 to 0 M urea in buffer containing 0.1 M Na-phosphate, pH 7.8, and 300 mM NaCl to facilitate the slow removal of the urea. The resin was then washed with a buffer containing 0.1 M Na-phosphate, pH 6.0, and 500 mM NaCl. The recombinant protein was eluted with a gradient of 0 –500 mM Imidazole in 0.1 mM Na-phosphate, pH 7.0, and 10% glycerol. The fractions were analyzed on SDS–PAGE and those containing the protein were pooled and dialyzed against T10E5 (Tris 10 mM and EDTA 5 mM, pH 8.0) buffer. The dialyzed sample was loaded onto the Mono-Q col- umn. The protein was eluted with a gradient of 0 to 1 M NaCl in T10E5. The rPA was dialyzed against 10 mM Hepes overnight and stored in aliquots at -70°C. |
| Refolding Assay | Binding assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |