Refolding Record:
| Protein | |
|---|---|
| Protein Name | Aspergillopepsin-2 |
| Abbreviated Name | n/a |
| SCOP Family | Peptidase A4 [101656] |
| Structure Notes | |
| Organism | Aspergillus niger |
| UniProt Accession | Unknown |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 213 |
| Molecular Weight | 22269.6 |
| Pi | 3.37 |
| Molecular Weight | 22269.6 |
| Disulphides | 2 |
| Full Sequence |
EEYSSNWAGAVLIGDGYTKVTGEFTVPSVSAGSSGSSGYQSEEYCASAWVGIDGDTCETAILQTGVDFCYEDGQTSYDAWYEWYPDYAYDFSDITISEGDSIKVTVEATSKSSGSATVENLTTGQSVTHTFSGNVEGDLCETNAEWIVEDFESGDSLVAFADFGSVTFTNAEATSGGSTVGPSDATVMDIEQDGSVLTETSVSGDSVTVTYV
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Huang XP, Kagami N, Inoue H, Kojima M, Kimura T, Makabe O, Suzuki K, Takahashi K. (2000) Biologycal Chemistry, 275, 26607-26614 |
| Project Aim | Expression and identification |
| Fusion | None |
| 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 | 150 min |
| Expression Vector | pAR-ANA |
| Expression Protocol | The sense and antisense primers contain a NdeI and BamHI site, respectively. The amplified DNA was digested with NdeI and BamHI and was inserted into the NdeI/BamHI site of pAR2113. Expression was carried out according to the method of Studier et al. (28). For preculture, 0.1 ml of a glycerol stock of transformed E. coli BL21 (DE3) with pAR-ANA was seeded into 10 ml of M9ZB broth (28) containing 0.2 mg/ml ampicillin. After shaking at 37 °C for a few hours, the culture became slightly turbid. It was then seeded into 1 liter of M9ZB broth containing 0.05 mg/ml ampicillin and incubated at 37 °C with shaking. When the absorbance at 600 nm reached 0.6-1.0, the expression of aspergillopepsinogen II was induced by the addition of isopropyl--D-thiogalactopyranoside (final concentration, 1 mM). The culture was further incubated at 37 °C for 150 min with shaking. The cells were harvested by centrifugation and suspended in 50 ml of a lysis buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride). The suspension was frozen in liquid nitrogen and then thawed. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6-1.0 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 8 M urea, 100 mM 2-mercaptoethanol, 50 mM NaCl, 1 mM EDTA, 50 mM sodium phosphate, pH 6.2 |
| Refolding Buffer | 50 mM sodium acetate, pH 5.25 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 5.25 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | overnight |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | To this was added lysozyme to a final concentration of 1 mg/ml, and the mixture was kept on ice for 30 min. The resulting solution was subjected to sonication (100 watts for 20 s, repeated four times) and then centrifuged at 17,000 × g for 10 min. After removing the supernatant, the pellet was dissolved in 50 ml of a denaturation buffer (8 M urea, 100 mM 2-mercaptoethanol, 50 mM NaCl, 1 mM EDTA, 50 mM sodium phosphate, pH 6.2). After standing at room temperature for 1 h, the solution was centrifuged at 100,000 × g for 30 min. The supernatant was loaded onto a DE52 (Whatman) column (1.5 × 6 cm) and was eluted with a linear gradient of 0.05-0.35 M NaCl. The fractions containing denatured aspergillopepsinogen II were pooled. The solution of denatured aspergillopepsinogen II was diluted with 10 volumes of a refolding buffer (50 mM sodium acetate, pH 5.25) and left to stand overnight at 4 °C. Then the solution was dialyzed against the refolding buffer at 4 °C for 24 h. |
| Refolding Assay | Proteolytic activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 80% |
| Purity | n/a |
| Notes | n/a |