Refolding Record:
| Protein | |
|---|---|
| Protein Name | Alpha-fetoprotein |
| Abbreviated Name | AFP |
| SCOP Family | Serum albumin-like |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P02771 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 619 |
| Molecular Weight | 68677.6 |
| Pi | 5.47927 |
| Molecular Weight | 68677.6 |
| Disulphides | 10 |
| Full Sequence |
MKWVESIFLIFLLNFTESRTLHRNEYGIASILDSYQCTAEISLADLATIFFAQFVQEATY
KEVSKMVKDALTAIEKPTGDEQSSGCLENQLPAFLEELCHEKEILEKYGHSDCCSQSEEG
RHNCFLAHKKPTPASIPLFQVPEPVTSCEAYEEDRETFMNKFIYEIARRHPFLYAPTILL
WAARYDKIIPSCCKAENAVECFQTKAATVTKELRESSLLNQHACAVMKNFGTRTFQAITV
TKLSQKFTKVNFTEIQKLVLDVAHVHEHCCRGDVLDCLQDGEKIMSYICSQQDTLSNKIT
ECCKLTTLERGQCIIHAENDEKPEGLSPNLNRFLGDRDFNQFSSGEKNIFLASFVHEYSR
RHPQLAVSVILRVAKGYQELLEKCFQTENPLECQDKGEEELQKYIQESQALAKRSCGLFQ
KLGEYYLQNAFLVAYTKKAPQLTSSELMAITRKMAATAATCCQLSEDKLLACGEGAADII
IGHLCIRHEMTPVNPGVGQCCTSSYANRRPCFSSLVVDETYVPPAFSDDKFIFHKDLCQA
QGVALQTMKQEFLINLVKQKPQITEEQLEAVIADFSGLLEKCCQGQEQEVCFAEEGQKLI
SKTRAALGV
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Boismenu R, Semeniuk D, Murgita RA. (1997) Protein Expression and Purification, 10, 10-26 |
| Project Aim | Structure-Function |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 30.0 |
| Expression Time | 10h |
| Expression Vector | pTrp4 |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | None |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 50mM Tris-HCl, 10mM EDTA, 0.2% Triton-X100, pH 8.0 |
| Solubilization Buffer | 0.1 M potassium phosphate, 6M guanidinium chloride, 0.1M betamercaptoethanol, pH 8.5 |
| Refolding Buffer | 50mM Tris-HCl, 100mM NaCl, 1mM EDTA |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 24h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | All procedures were carried out at 47C. Each frozen pellet from a 1-liter culture of E. coli expressing AFP was resuspended in 25 ml of lysis buffer A [50 mM Tris-HCl (pH 7.5), 20% sucrose, 100 mg/ml lysozyme, 10 mg/ml DNase, 10 mg/ml RNase, 50 mg/ml PMSF] and incubated for 10 min. EDTA was added to a final concentration of 35 mM, and the extracts allowed to stand a further 10 min. Following the addition of 25 ml of lysis buffer B [50 mM Tris-HCL (pH 7.5), 25 mM EDTA, 0.2% Triton X-100], the lysates were incubated an additional 30 min. The cell lysates were centrifuged at 12,000g for 20 min, and the inclusion bodies containing the rAFPs were washed twice with 50 ml of wash buffer [50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.2% Triton X-100], followed each time by centrifugation as above. The inclusion bodies containing rAFPs were solubi lized and refolded using a procedure modified from that of Latta et al. (26). Briefly, inclusion bodies were dissolved in 50 ml of denaturation buffer [0.1 M K2HPO4 pH 8.5), 6 M guanidine-HCl, 0.1 M 2-mercaptoethanol], and mixed for 18 h at 47C. The solubilized extract was diluted 50-fold in 50 mM Tris-HCl (pH 8.5), 100 mM NaCl, 1mM EDTA, and the rAFP proteins were allowed to renature for at least 24 h at 47C. Renatured rAFPs were concentrated using an Amicon S1Y30 (MW cutoff 39 kDa; Amicon, Beverly, MA) or S1Y10 (MW cutoff 10 kDa) spiral membrane cartridge-equipped system. The final solution was clarified through a Millex 0.22-mm membrane filter (Millipore) and dialyzed against phosphate-buffered saline (PBS). |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 65% |
| Purity | 40% |
| Notes | |