Refolding Record:
| Protein | |
|---|---|
| Protein Name | Apolipoprotein(a)(lipoprotein kringle) |
| Abbreviated Name | LK68 |
| SCOP Family | Ricin B-like |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P08519 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | n |
| Domain | apo(a)KIV37 domain composed of 92 amino acid |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 114 |
| Molecular Weight | 12756.1 |
| Pi | 5.83 |
| Molecular Weight | 12756.1 |
| Disulphides | 4 |
| Full Sequence |
APTEQTPVVRQCYHGNGQSYRGTFSTTVTGRTCQSWSSMTPHRHQRTPENYPNDGLTMNYCRNPDADTGPWCFTMDPSIRWEYCNLTRCSDTEGTVVAPPTVIQVPSLGPPSEQ
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | LoGrasso PV, Cornell-Kennon S, Boettcher BR. (1994) Biologycal Chemistry, 26, 21820-21827 |
| Project Aim | Identification and 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) |
| Expression Temp | 37.0 |
| Expression Time | 3 h |
| Expression Vector | PET14b |
| Expression Protocol | Expression of Fusion Apo(a) KN37-The enzyme was expressed in the E. coli strain BL21(DE3) pLysS (Rosenberg et al., 1987) under the following conditions: 1 liter of Luna Broth (10 g of tryptone (Difco), 5 g of yeast extract (Difco), 5 g of NaCVliter of distilled H,O, pH raised to 7.4) containing 200 pg/ml ampicillin (Sigma) and 30 pg/ml chloramphenicol (Sigma) was inoculated with 1 ml of a saturated culture of BL2UDE3) pLysS harboring the pET14b apo(a) KIV37 plasmid and grown at 37 \"C with agitation at 220 rpm until the culture achieved an A, nm = 0.4-0.6. At this point, cultures were induced by the addition of isopropylthio-P-o-galactoside (IPTG) (Life Technologies, Inc.) at a final concentration of 0.4 mM. Cells were grown an additional 3 h after induction with typical AGOOnm readings reaching approximately 1.9. Cells were harvested by centrifugation at 6000 x g for 10 min at 4 \"C, and cell pellets were frozen at -70 \"C. In addition, 1-ml aliquots were taken from the induced culture at 0 h (i.e. immediately prior to induction) and at 1,2, and 3 h following induction. These cell pellets were frozen at -20 \"C for use in Tricine-PAGE analysis. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.4-0.6 = 600 |
| Cell Disruption Method | Freeze-thaw |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 15 ml of 50 mM Tris, pH 8.0,l mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 uM leupeptin, 1 UM pepstatin, 0.25 mM paminoethylbenzenesulfonyl fluoride (AEBSF), and 200 ul of 5 mg/ml DNase I |
| Solubilization Buffer | 3 ml of 100 mM Tris, pH 8.0, containing 6 M guanidine HCl, 1 mM EDTA, and 0.1 M dithiothreitol |
| Refolding Buffer | 100 mM Tris, pH 8.0, containing 0.4 M arginine, 1 mM EDTA, and 4 mM oxidized glutathione |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 10.0 |
| Protein Concentration | n/a |
| Refolding Time | 5 days |
| Redox Agent | GSSG |
| Redox Agent Concentration | 4 mM |
| Refolding Protocol | Cell Fractionation and Refolding of Fusion Apo(a) KN37-E. coli cells were lysed using a combination of a freeze-thaw cycle and cell wall degradation by lysozyme, which is expressed from the pLysS plasmid during cell growth and released upon reeze-thaw. The cell pellet from one liter of E. coli culture (2.2 g of cell paste, wet weight) was resuspended in 15 ml of 50 mM Tris, pH 8.0,l mM EDTA, 1 mM phenylmethylsulfonyl fluoride (Sigma), 10 uM leupeptin (Sigma), 1 uM pepstatin (Sigma), 0.25 mM p-aminoethylbenzenesulfonyl fluoride (AEBSF) (Boehringer Mannheim), and 200 pl of 5 mg/ml DNase I (Sigma). This suspension was gently stirred at 4 \"C for 3045 min until the solution was no longer viscous. The suspension was centrifuged at 17,000 x g for 45 min at 4 \"C and the pellet used for subsequent purification. Refolding of proteins in the insoluble cell debris fraction was patterned after Buchner and Rudolph (1991). The pellet from above was resuspended in 3 ml of 100 mM Tris, pH 8.0, containing 6 M guanidine HCl (Sigma), 1 mM EDTA, and 0.1 M dithiothreitol (U. S. Biochemical Corp.), and stirred at room temperature for 2 h. This solution was diluted 33-fold into 100 mM Tris, pH 8.0, containing 0.4 M arginine (Sigma), 1 mM EDTA, and 4 mM oxidized glutathione (Sigma). This sample was incubated at 10 \"C for 5 days. The solution was clarified by centrifugation at 17,000 x g for 30 min at 4 \"C, and the supernatant was saved as the refolded fusion apo(a) KIV37 fraction. The sample was concentrated on a YM3 membrane (Amicon) to approximately 50 ml and subsequently dialyzed for 20 h against three 1-liter changes of 50 mM Tris, 1 mM EDTA, pH 8.0. |
| Refolding Assay | Unspecified |
| Refolding Chaperones | None |
| Refolding Additives | L-Arginine |
| Additives Concentration | 0.4 M |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |