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 | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 329 |
| Molecular Weight | 36696.7 |
| Pi | 6.15 |
| Molecular Weight | 36696.7 |
| Disulphides | Unknown |
| Full Sequence |
APPEKSPVVQDCYHGDGRSYRGISSTTVTGRTCQSWSSMIPHWHQRTPENYPNAGLTENYCRNPDSGKQPWCYTTDPCVRWEYCNLTQCSETESGVLETPTVVPVPSMEAHSEAAPTEQTPVVRQCYHGNGQSYRGTFSTTVTGRTCQSWSSMTPHRHQRTPENYPNDGLTMNYCRNPDADTGPWCFTMDPSIRWEYCNLTRCSDTEGTVVAPPTVIQVPSLGPPSEQDCMFGNGKGYRGKKATTVTGTPCQEWAAQEPHRHSTFIPGTNKWAGLEKNYCRNPDGDINGPWCYTMNPRKLFDYCDIPLCASSSFDCGKPQVEPKKCPGS
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Choi WC, Kim MY, Suh CW, Lee EK (2005) Process Biochemistry, 40, 1967-1972 |
| Project Aim | Protein refolding |
| 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 | 0.0 |
| Expression Time | 00 |
| Expression Vector | pETIIa |
| Expression Protocol | The recombinant E. coli strain (BL21 (DE3)) equipped with pET IIa vector system and the LK68 reference standard were supplied by Mogam Biotechnology Research Institute (YongIn, Korea.) Approximately 25% of the total proteins expressed were LK68 (pI 6.1, 37 kDa). The E. coli culture broth was centrifuged at 10,000 rpm for 20 min for cell harvesting and the washed cells were disrupted in a ‘cell disruption buffer’ using an ultrasonic disintegrator (550 Sonic Dismembrator, Fisher, USA). Dynamic light scattering (LPA-300, Otsuka, Japan) was used to measure particulate sizes of cell debris from the cell rupture step. |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | ultrasonication |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding-Expanded bed adsorption chromatography (EBA) |
| Wash Buffer | 20 mM Tris–HCl, 50 mM deoxycholic acid (pH 7.5) |
| Solubilization Buffer | 8 M urea, 20 mM Tris–HCl, 100 mM DTT (pH 9.0) |
| Refolding Buffer | 20 mM Tris–HCl (pH 9.0) |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no tag |
| Refolding pH | 9.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | EBA-mediated refolding Twenty-five millilitre of the dissolved LK68 inclusion body (2.5 mg/mL) was loaded to STREAMLINE 25 column (Amersham Biosciences, Uppsala, Sweden) that was filled with 50 mL STREAMLINE DEAE resin (Amersham Biosciences). The bed expansion ratio was 3.0. Entrapped debris was washed with four column volumes of the ‘column-washing buffer’ and then by 10 column volumes of the ‘refolding buffer’ to remove urea gradually and initiate solid-phase refolding. After settling the resin, the ‘elution buffer’ was fed from top to bottom in a step-wise gradient (10, 20, 30, 40, and 100%) to elute the refolded protein. The column was regenerated with ‘regeneration buffer’ [7]. The same procedure was applied for the solid-phase refolding from cell homogenate. |
| Refolding Assay | RP-HPLC,SEC-HPLC |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 44% |
| Purity | n/a |
| Notes | n/a |