Refolding Record:
| Protein | |
|---|---|
| Protein Name | C-type lectin also known as asialofetuin-binding C-type` |
| Abbreviated Name | OLABL |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Spirinchus lanceolatus |
| UniProt Accession | Q3LHY4 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 138 |
| Molecular Weight | 15490.0 |
| Pi | 5.84 |
| Molecular Weight | 15490.0 |
| Disulphides | Unknown |
| Full Sequence |
SYPSCPSRQWTKNGQRCYLSVSAPNNWVGAEQYCLRQGANLASVHSFSEYTFLQQLVGSESNGHPVTWIGGTDAFQDRVWFWSDGSSFDYAAWAAGEPNNYGGRREPCIEMNWGADHRWNDSPCDNKRGFICSFKLC
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hosono M, Sugawara S, Ogawa Y, Kohno T, Takayanagi M, Nitta K. (2005) Biochemica et Biophysica Acta, 1725, 160-173 |
| Project Aim | Purification & characterization |
| 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)pLysS |
| Expression Temp | 37.0 |
| Expression Time | 6 h |
| Expression Vector | pET16L |
| Expression Protocol | To construct the expression vector of OLABL, NdeI and BamHI restriction sites were introduced to both cDNAs by PCR amplification using linker primers, ExF1 and ExR1. The DNA was ligated into NdeI- and BamHI-digested pET16 vector (Novagen, Darmstadt, Germany) using T4 DNA ligase (New England Biolabs Ltd., Pickering, Canada). The host Escherichia coli strain, BL21(DE3) pLysS (Novagen), was transformed with recombinant plasmids. Positive transfectants (pET16-H and pET16-L) were identified by direct PCR and restriction enzyme digestion. BL21(DE3) pLysS cells transformed with pET16-H and pET16-L were grown at 37 °C in Luria-Bertani (LB) medium containing 50 μg/mL carbenicillin and 34 μg/mL chloramphenicol until the optical density at 600 nm reached 0.5–0.6. Then, isopropyl 1-thio-β-d-galactoside (IPTG) was added to the culture medium at a final concentration of 1 mM, and cultivation was continued for an additional 6 h at 37 °C. Cells were collected by centrifugation at 7200 rpm for 15 min, frozen at −80 °C, and subsequently thawed to disrupt cell walls. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.5-0.6 = 600 |
| Cell Disruption Method | Freeze-thaw |
| Lytic Agent | None |
| Pre-Refolding Purification | Ni-NTA column |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 20 mM PB containing 8 M urea) at pH 6.3, 5.9, and 4.5 |
| Solubilization Buffer | 20 mM PB containing 6 M guanidine hydrochloride and 0.5 mM NaCl, pH 7.8 |
| Refolding Buffer | 250 mM imidazole was diluted with a washing buffer (pH 8.0) containing 0.2 mM 2-ME |
| Pre-Refolding Purification | Ni-NTA column |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 48 |
| Redox Agent | Beta-mercaptoethanol |
| Redox Agent Concentration | 0.2 mM |
| Refolding Protocol | Cells were collected by centrifugation at 7200 rpm for 15 min, frozen at −80 °C, and subsequently thawed to disrupt cell walls. The cells were suspended in guanidine lysis buffer (20 mM PB containing 6 M guanidine hydrochloride and 0.5 mM NaCl, pH 7.8), agitated for 20 min, and sonicated for 30 s. The lysate was centrifuged at 15,000 rpm for 30 min at 4 °C, and the supernatant was loaded on nickel-nitrilotriacetic acid His bind resin (0.5 mL of resin for every 100 mL starting culture). The resin was washed successively with washing buffer (20 mM PB containing 8 M urea) at pH 6.3, 5.9, and 4.5. The adsorbed protein was eluted with washing buffer at pH 5.9, increasing the molarity of imidazole (20, 50, 100, and 250 mM). Each of the denatured proteins was refolded following the procedure of Ewart et al. [29] with modifications. Briefly, the protein eluted with washing buffer containing 250 mM imidazole was diluted with a washing buffer (pH 8.0) containing 0.2 mM 2-ME until the final protein concentration was about 100 μg/mL. The solution was dialyzed against 50 mM Tris–HCl (pH 8.0) containing 100 mM NaCl and 1 mM CaCl2 for 48 h, and subsequently against 100 mM NH4HCO3 for 72 h at 4 °C. The resulting solution was evaporated in vacuo and redissolved in distilled water. This His-tagged subunit was treated with factor Xa at 20 °C for 16 h. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |