Refolding Record:
| Protein | |
|---|---|
| Protein Name | Bone morphogenetic protein 3 |
| Abbreviated Name | BMP3 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Rat |
| UniProt Accession | P49002 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 112 |
| Molecular Weight | 12381.4 |
| Pi | 8.21 |
| Molecular Weight | 12381.4 |
| Disulphides | 4 |
| Full Sequence |
QWIEPRNCARRYLKVDFADIGWSEWIISPKSFDAYYCSGACQFPMPKSLKPSNHATIQSIVRAVGVVSGIPEPCCVPEKMSSLSILFFDENKNVVLKVYPNMTVDSCACR
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Han B, Perelman N, Tang B, Hall F, Shors EC, Nimni ME. (2002) J Orthop Res., 20, 747-755 |
| Project Aim | Diagnostic studies |
| 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 | 5 h |
| Expression Vector | pET28b |
| Expression Protocol | nant BMP3 fusion proteins bearing a purification tag, as well as an auxillary ECM-binding domain, which were then purified and refoldedkenatured in vitro to reconstitute their native biologically active dimer conformaThe PCR amplified cDNA fragment was purified from an agarose gel using the Geneclean kit (BiolOl, Carsbas, CA), inserted into a high-performance bacterial expression vector. pET28b (Novagen, Madison. WI), and transformed into the BL21(DE), strain of E. coli. Protein expression was induced with 0.4 mM isopropyl P-D-thioga-lactopyranoside (IPTG) at 37 \"C for 5 h. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | Ni-NTA column |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 8 M urea containing the proteinase inhibitor, 10 mM phenylmethysulfonyl flu |
| Refolding Buffer | lowering the concentration of urea (1.5-2.0 M) 0.2 mM of oxidized glutathione (GSSG) and 2.0 mM of reduced glutathione (GSH) |
| Pre-Refolding Purification | Ni-NTA column |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 24 |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 2/0.2 mM |
| Refolding Protocol | The recombinant proteins, which accumulated in the insoluble inclusion bodies, were isolated from the cell lysate by centrifugation and solubilized with 8 M urea containing the proteinase inhibitor, 10 mM phenylmethysulfonyl fluoride (PMSF). The solubilized rhBMP3-C was purified from extraneous bacterial proteins under denaturing conditions by Ni-NTA chelating chromatography (Qiagen, Valencia, CA). Purified rhBMP3- C monomers were gradually renatured over 24 h at 4 \"C by rapidly lowering the concentration of urea (1.5-2.0 M) and employing a controlled glutathione redox system, which includes 0.2 mM of oxidized glutathione (GSSG) and 2.0 mM of reduced glutathione (GSH). The refolding procedure was completed by stepwise dialysis into a 10%1 sucrose phosphate buffer (0.01 M, pH 8.0). and the renatured fusion proteins were stored at -20 \"C prior to use. Purity and yields of recombinant proteins were analyzed by electrophoresis on an 8 16% SDS-PAGE gel. A recombinant BMP3 fusion protein lacking the collagen-targeting domain but containing the polyhistidine purification sequences (rhBMP3) was also prepared and expressed using the same approach to serve as a control. |
| Refolding Assay | Binding assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |