Refolding Record:
| Protein | |
|---|---|
| Protein Name | Protein p1 |
| Abbreviated Name | gp1 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Bacteriophage phi-29 |
| UniProt Accession | Q6LDQ1 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 86 |
| Molecular Weight | 9892.5 |
| Pi | 7.95 |
| Molecular Weight | 9892.5 |
| Disulphides | Unknown |
| Full Sequence |
MGKIFDQEKRLEGTWKNSKWGNQGIIAPVDGDLKMIDLELEKKMTKLEHENKLMKNALYELSRMENNDYATWVIKVLFGGAPHGAK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hashiyama K, Takeuchi A, Makino O. (2005) Biochem Biophys Res Commun., 331, 1310-1316 |
| Project Aim | Undefined |
| Fusion | C-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 | 2 h |
| Expression Vector | pUV1-WT |
| Expression Protocol | Purification of wild type gp1 or variants. The products of gene 1 of wild type or variants were purified as described [9] with some modifications. The recombinant gp1 in E. coli DH5 containing each plasmid was induced with IPTG. After induction for 2 h at 37 °C, cells were collected by centrifugation. After cell disruption, the bacterial lysate was separated into soluble and insoluble fractions by centrifugation (10,000g, 15 min, 4 °C). To determine the distribution of gp1 in the soluble or insoluble fractions, aliquots from each fraction were subjected to 13% SDS–PAGE and the gel was stained with CBB [16]. Fractions, either soluble (W71R and K74E) or insoluble (K2E, K8E, K33E, K41E, and F77S), which contained the majority of gp1 were solubilized by denaturing buffer containing 6 M guanidium–HCl, 100 mM sodium phosphate, and 10 mM imidazole at pH 7.4. Each gp1 was further purified by using a Ni column (HiTrap chelating) according to the method suggested by the manufacturer (Amersham Biosciences). The eluate in elution buffer (6 M guanidium–HCl, 100 mM sodium phosphate, and 300 mM imidazole at pH 7.4) was collected and analyzed by 13% SDS–PAGE. Fractions containing gp1 were separated into aliquots and stored at −80 °C until refolding. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Not stated |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | partial |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 6 M guanidium–HCl, 100 mM sodium phosphate, and 10 mM imidazole at pH 7.4. |
| Refolding Buffer | 1 M urea, 50 mM Tris–HCl (pH 8.0), 1 M NaCl, 0.5% Chaps , and 0.1 mM EDTA and without urea |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 17.5 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Refolding of gp1. To refold the denaturated gp1 and to remove high concentrations of imidazole and guanidium–HCl, the stored eluate from Ni column was dialyzed against 125 volumes of refolding buffer containing 1 M urea, 50 mM Tris–HCl (pH 8.0), 1 M NaCl, 0.5% Chaps (Dojindo Laboratories), and 0.1 mM EDTA at 4 °C for 16 h followed by dialysis against 125 volumes of refolding buffer without urea (20 mM Tris–HCl, pH 8.0, 1 M NaCl, 0.5% Chaps, and 0.1 mM EDTA) at 4 °C for 1.5 h twice. |
| Refolding Assay | Activity assay |
| Refolding Chaperones | None |
| Refolding Additives | CHAPS |
| Additives Concentration | 0.5% |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |