Refolding Record:
| Protein | |
|---|---|
| Protein Name | Extracellular zinc metalloprotease |
| Abbreviated Name | ZmpA |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Burkholderia cepacia KWI-56 |
| UniProt Accession | Q2VL24 |
| 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 | 542 |
| Molecular Weight | 57347.4 |
| Pi | 6.9 |
| Molecular Weight | 57347.4 |
| Disulphides | Unknown |
| Full Sequence |
GDQPAAVDKALQLIQQNPSAFSLAAGGTARTLKFAGPQAGAPTDGDQFQVRDVIVDPDGTEHVRFDRFHAGLPVIGGDVVVHSSKGQLKQASVTQLAPINLAGSIGKVGNRAVIRNAPDVGATRARHIAAARFNSDVRRVDDAELVVFARDVTPTLAYAVRVYGKATETHGDAVLYYVDARTGTLLDAQDLIKTAAATGTGRSLYYGNLTLTTDQTGTNAYRMLDPSRGGGSVYDGRGLSSDEVEQATDLPIFTSSTNVWGNNATTDRQTVAADIDYGLALTWDYYKTTHNRNGIFNDGRGVKSYAHVVFNTGSGTTGANAAWLSSRVMAYGDGVPGTRLPKPVVSVDVAGHEMSHGVTEATANLNYSGDAGGLNESTSDIFGTLVKFYANNPNDPGNYVIGARVVSGGLRKMYKQDLDGRSFSCYPSGGFSWSNPRHDPHFTSGVGNRFFYLLSEGPSVPPTDTGLSRTQLVCNGDTTFSGLGRDKAGKIWYRTLTVYLNANSSYPNARRASIQAANDLYGANSAESATVARAWSAVGVN
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Kooi C, Corbett CR, Sokol PA. (2005) J Bacteriol., 187, 4421-4429 |
| Project Aim | Analysis |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | DH5α |
| Expression Temp | 37.0 |
| Expression Time | 3.5 h |
| Expression Vector | pCK10 |
| Expression Protocol | E. coli DH5 pCK10 subcultures were induced with 0.6 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) at an optical density at 600 nm (OD600) of approximately 0.4. The optimum incubation time was determined by removing 1 ml of the culture at 1, 2, 3, and 4 h after induction and analyzing the normalized samples by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), after which cultures were typically harvested after 3.5 h of induction. Cultures were centrifuged at 8,000 x g for 20 min. Pellets were resuspended in 10 ml sonication buffer (SB; 50 mM sodium phosphate, pH 8.0, 300 mM NaCl), sonicated for 5 15-s bursts and centrifuged at 15,000 x g to determine if the expressed ZmpA was soluble or insoluble. The insoluble pellets were washed stepwise with SB and 2 M sodium chloride, SB and 0.25% Triton X-100, and SB and 2 M sodium chloride. The samples were centrifuged at 6,000 x g between washes. The final pellets were resuspended in SB with 6 M guanidine hydrochloride. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.4 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: affinity immobilization |
| Wash Buffer | 2 M sodium chloride, SB and 0.25% Triton X-100, and SB and 2 M sodium chloride |
| Solubilization Buffer | 6 M guanidine hydrochloride |
| Refolding Buffer | 50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 6 M guanidine hydrochloride |
| Pre-Refolding Purification | None |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 1 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Refolding of recombinant mature ZmpA and preproZmpA. The solubilized mature ZmpA or preproZmpA was bound to Ni-nitrilotriacetic acid (NTA) (QIAGEN), which had been preequilibrated in SB, containing 6 M guanidine hydrochloride, for at least 1 h at 4°C. The bound ZmpA was washed with 5 column volumes (CV) of 20 mM Tris-HCl, pH 7.4, 6 M guanidine hydrochloride (buffer A), and 5 CV 50 mM Tris-HCl, pH 7.4, 20% glycerol, 500 mM sodium chloride, and 6 M urea (buffer B), followed by 50 CV buffer B to 50 mM Tris-HCl, pH 7.4, 20% glycerol, 500 mM sodium chloride, 1 M urea (buffer C) linear gradient. The bound ZmpA was washed with 5 CV buffer C, 5 CV 50 mM Tris-HCl, pH 7.4, 10% glycerol, 50 mM NaCl (buffer D), and 5 CV SB. The refolded ZmpA was eluted from the Ni-NTA with 100 mM imidazole. The eluted ZmpA was typically dialyzed against 10 mM Tris-HCl, pH 7.2, prior to proteolytic analysis of the enzyme. Proteolytic activity was determined using 7.5 mg hide powder azure (Sigma-Aldrich) in 1.5 ml 10 mM Tris-HCl (43). One unit of activity was defined as the amount of protease required to cause a change in the OD595 of 0.1 in 1 h (43). |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |