Refolding Record:
| Protein | |
|---|---|
| Protein Name | DNA Polymerase Delta |
| Abbreviated Name | DNA Polymerase Delta |
| SCOP Family | DNA polymerase type-B |
| Structure Notes | |
| Organism | Yeast (Saccharomyces cerevisiae) |
| UniProt Accession | P15436 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Heterodimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 1115 |
| Molecular Weight | 124591.0 |
| Pi | 8.23383 |
| Molecular Weight | 124591.0 |
| Disulphides | 0 |
| Full Sequence |
MSEKRSLPMVDVKIDDEDTPQLEKKIKRQSIDHGVGSEPVSTIEIIPSDSFRKYNSQGFK
AKDTDLMGTQLESTFEQDVSQMEHDMADQEEHDLSSFERKKLPTDFDPSLYDISFQQIDA
EQSVLNGIKDENTSTVVRFFGVTSEGHSVLCNVTGFKNYLYVPAPNSSDANDQEQINKFV
HYLNETFDHAIDSIEVVSKQSIWGYSGDTKLPFWKIYVTYPHMVNKLRTAFERGHLSFNS
WFSNGTTTYDNIAYTLRLMVDCGIVGMSWITLPKGKYSMIEPNNRVSSCQLEVSINYRNL
IAHPAEGDWSHTAPLRIMSFDIECAGRIGVFPEPEYDPVIQIANVVSIAGAKKPFIRNVF
TLNTCSPITGSMIFSHATEEEMLSNWRNFIIKVDPDVIIGYNTTNFDIPYLLNRAKALKV
NDFPYFGRLKTVKQEIKESVFSSKAYGTRETKNVNIDGRLQLDLLQFIQREYKLRSYTLN
AVSAHFLGEQKEDVHYSIISDLQNGDSETRRRLAVYCLKDAYLPLRLMEKLMALVNYTEM
ARVTGVPFSYLLARGQQIKVVSQLFRKCLEIDTVIPNMQSQASDDQYEGATVIEPIRGYY
DVPIATLDFNSLYPSIMMAHNLCYTTLCNKATVERLNLKIDEDYVITPNGDYFVTTKRRR
GILPIILDELISARKRAKKDLRDEKDPFKRDVLNGRQLALKISANSVYGFTGATVGKLPC
LAISSSVTAYGRTMILKTKTAVQEKYCIKNGYKHDAVVVYGDTDSVMVKFGTTDLKEAMD
LGTEAAKYVSTLFKHPINLEFEKAYFPYLLINKKRYAGLFWTNPDKFDKLDQKGLASVRR
DSCSLVSIVMNKVLKKILIERNVDGALAFVRETINDILHNRVDISKLIISKTLAPNYTNP
QPHAVLAERMKRREGVGPNVGDRVDYVIIGGNDKLYNRAEDPLFVLENNIQVDSRYYLTN
QLQNPIISIVAPIIGDKQANGMFVVKSIKINTGSQKGGLMSFIKKVEACKSCKGPLRKGE
GPLCSNCLARSGELYIKALYDVRDLEEKYSRLWTQCQRCAGNLHSEVLCSNKNCDIFYMR
VKVKKELQEKVEQLSKW
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Brown WC, Duncan JA, Campbell JL. (1993) J Biol Chem, 268, 982-90 |
| Project Aim | Undefined |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | HMS174 |
| Expression Temp | 37.0 |
| Expression Time | unknown |
| Expression Vector | unknown |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Solubility | soluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | unknown |
| Solubilization Buffer | 25mM Tris-HCl, 25mM NaCl, 1mM DTT, 6M urea, pH 8.0 |
| Refolding Buffer | 50mM Tris-HCl, 50mM NaCl, 10% glycerol |
| Pre-Refolding Purification | Ion-exchange chromatography |
| Tag Cleaved | yes |
| Refolding pH | 7.4 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 5 micrograms/ml |
| Refolding Time | 28h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The 0.8 M salt fraction from the purification of full-length DNA polymerase 6 and the flow-through and high salt fraction of the truncated form were diluted to 5 microgram/ml for renaturation. These were then dialyzed sequentially against the following buffers maintaining a ratio of no greater than 50 for dialysis volume to sample volume. The dilution buffer is 0.2 M Tris-HC1 (pH 8.0), 0.5 M NaC1, 1 mM DDT, and 6 M urea. The samples were first dialyzed against 0.2 M Tris-HCI (pH 8.0), 0.5 M NaCI, 1 mM DTT, 100 pM ZnCl, 2 mM MgCl,, and 4 M urea for at least 8 h at 4 C. The next three dialysis steps employ buffers that contain the same DTT, ZnCl, and MgCl, concentration but vary in Tris, NaCI, and urea concentrations. The second buffer contains 0.1 M Tris-HCI (pH 8.0), 0.5 M NaCI, and 2 M urea, whereas the third contains 50 mM Tris-HCI (pH 7.4), 0.2 M NaCl, and 1 M urea. The final buffer had 50 mM Tris-HC1 (pH 7.4), 50 mM NaCI, 10% glycerol and did not contain urea. The final dialysis was stopped after 4 h. Samples were then either stored on ice or concentrated on a 0.1-ml DE52 column using 50 mM Tris-HC1 (pH 7.4), 0.45 M NaC1, 1 mM DTT, 10% glycerol, and 50% ethylene glycol to elute bound polymerase. These samples were then frozen in dry ice/ethanol and stored at -70 C |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | |
| Purity | |
| Notes | |