Refolding Record:
| Protein | |
|---|---|
| Protein Name | DNA polymerase beta |
| Abbreviated Name | Polbeta |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Leishmania infantum |
| UniProt Accession | Q9U6N3 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 376 |
| Molecular Weight | 42700.3 |
| Pi | 9.08 |
| Molecular Weight | 42700.3 |
| Disulphides | Unknown |
| Full Sequence |
MLRRKFLRRDHRENIIRIFQEMADLNNALGEKYKVSSYHRSIESLKTNLDKPLNTPQDLKAFSGFGAKLLKKAEEIMATGKLEELESKTKPKLKAIQELTQVHGFGPRAAAALFDREGIFTVDELLQKADSIPSLTDQQRVGIKYFYDINEKIPMQESVLHENYLREKCMEVLGKDFSILICGSYRRRHPFSGDVDAILSRTLDAPPLSEPVAATGVLGHFVEFLESLKYLEATMAQGPLKYMGMGRLPPRIVRDKAGRENTKVYKARRVDIRLIETKSVPTAMLTFTGSKNFNVIMRQAAISKGYLLNEYGLFKLGTPEEARALYERIGIRGKNAGEELGVPKDELEDKRVEVRSEQDVFDVLGMPYAKPENRDP
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Alonso A, Terrados G, Picher AJ, Giraldo R, Blanco L, Larraga V. (2006) DNA Repair, 5, 89-101 |
| Project Aim | Undefined |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | M15 |
| Expression Temp | 37.0 |
| Expression Time | 2 h |
| Expression Vector | pREP4 |
| Expression Protocol | The complete encoding sequence corresponding to Li Polβ (GenBank accession number AF182167) was cloned in the expression vector pQE32 (Qiagen, Germany), which allows for the expression of recombinant proteins as fusions to a multifunctional leader peptide containing a hexahistidyl sequence for purification on Ni2+-affinity resins. The expression of Li Polβ was carried out in the Escherichia coli strain M15 (pREP4) (Qiagen, Germany), induced by the addition of 0.1 mM isopropyl-β-d-thiogalactopyranoside (IPTG), and grown at 37 °C in LB plus ampicillin (100 μg/ml) and kanamycin (25 μg/ml) for 2 h. The cultured cells were harvested by centrifugation at 5000 rpm for 15 min at 4 °C, in a Sorvall GS3 rotor and washed in 50 ml of cold 150 mM NaCl. Cell pellets were resuspended in 50 ml of lysis solution (1 M KCl, 60 mM imidazole–HCl pH 8.0, 1% Brij-58, 1 mM p-NH2-benzamidine, 10% glycerol) and then disrupted using a MSE sonicator in an ice bath. Cell debris was separated from the soluble lysates by ultracentrifugation (17,500 rpm in a Beckman Ti 45 rotor, for 30 min at 4 °C). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | not specified |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 5 M guanidium hydrochloride (GuHCl) and 20 mM imidazole–HCl pH 8.0 |
| Refolding Buffer | 0.5 M (NH4)2SO4, 50 mM NH4-acetate pH 6.0, 1 mM ME, 10% glycerol |
| Pre-Refolding Purification | not specified |
| Tag Cleaved | no |
| Refolding pH | 6.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | 24 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The protein, aggregated as inclusion bodies, was solubilized by means of a modification of a protocol described to refold the DNA replication initiator RepA [30]. Inclusion bodies were thus solubilized by 1 min sonication in 5 M guanidium hydrochloride (GuHCl) and 20 mM imidazole–HCl pH 8.0. Remaining particles were removed by ultracentrifugation (15,000 rpm in a Beckman Ti 45 rotor, for 30 min at 4 °C) and the soluble fraction was purified by Ni-IMAC at 4 °C. A 10 ml Chelating Sepharose Fast-Flow column, preactivated with NiCl2, was equilibrated with at least 10 volumes of solution A (5 M GuHCl, 20 mM imidazole–HCl, 20 mM Hepes pH 8.0). Soluble lysate was then loaded at 1.0 ml/min flow rate into the column. This was extensively washed with solution A and then a linear 250 ml gradient was run from 0–100% of solution B (5 M GuHCl, 150 mM imidazole–HCl pH 8.0) at 1.0 ml/min. Fractions containing Li Polβ were identified by sodium dodecyl sulphate (SDS)-PAGE and pooled. Then, 2-mercaptoethanol (ME) was added to 10 mM and the sample was stored in 1 ml aliquots at −80 °C up to several months. These fractions were diluted to approximately 0.1 mg/ml (final volume 20 ml) with 5 M GuHCl, 0.5 M (NH4)2SO4, 200 mM NH4-acetate pH 6.0, 1 mM ME, 1.2% CHAPS (Sigma). Protein refolding was carried out by dialysis at 4 °C, against 3 × 1 l of 0.5 M (NH4)2SO4, 50 mM NH4-acetate pH 6.0, 1 mM ME, 10% glycerol for a total time of 24 h. Aggregated material was then removed by ultracentrifugation (37,500 rpm in a Beckman Ti45 rotor, for 60 min at 4 °C) and the soluble fraction concentrated in an Amicon 50 ml filtration cell (kept in an ice bath) with a Diaflo PM-10 membrane. Concentration of the purified fraction (0.8 mg/ml) was determined by measuring A280 in 5 M GuHCl, considering a molar extinction coefficient of 17,760 M−1 cm−1. DNA polymerase specific activity of the refolded soluble fraction was estimated as described below. |
| Refolding Assay | Activity assay |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol,CHAPS |
| Additives Concentration | 1.2%-10% |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |