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 | Dilution |
| 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 | 16 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a,n/a |
| Refolding Protocol | To obtain control data for refolding yield, the conventional, solution-phase refolding was carried out with the dissolved inclusion body by applying ‘simple dilution’ and ‘rapid dilution’ method. The simple dilution method was to add the ‘refolding buffer’ slowly to the dissolved LK68 solution, while the rapid dilution method was to add the protein solution to the ‘refolding buffer’. In both methods, the refolding proceeded for 16 h at pH 9.0. |
| Refolding Assay | RP-HPLC,SEC-HPLC |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 34-38% |
| Purity | n/a |
| Notes | refolding method were simple dilution with the yield of 38% and rapid dilution with the yield of 34% |