Refolding Record:
| Protein | |
|---|---|
| Protein Name | Penton base protein |
| Abbreviated Name | Penton base |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Adenovirus Type 2 |
| UniProt Accession | P03276 |
| 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 | 575 |
| Molecular Weight | 63559.1 |
| Pi | 5.24664 |
| Molecular Weight | 63559.1 |
| Disulphides | 0 |
| Full Sequence |
MQRAAMYEEG PPPSYESVVS AAPVAAALGS PFDAPLDPPF VPPRYLRPTG GRNSIRYSEL APLFDTTRVY LVDNKSTDVA SLNYQNDHSN FLTTVIQNND YSPGEASTQT INLDDRSHWG GDLKTILHTN MPNVNEFMFT NKFKARVMVS RSLTKDKQVE LKYEWVEFTL PEGNYSETMT IDLMNNAIVE HYLKVGRQNG
VLESDIGVKF DTRNFRLGFD PVTGLVMPGV YTNEAFHPDI ILLPGCGVDF THSRLSNLLG IRKRQPFQEG FRITYDDLEG GNIPALLDVD AYQASLKDDT EQGGDGAGGG NNSGSGAEEN SNAAAAAMQP VEDMNDHAIR GDTFATRAEE KRAEAEAAAE AAAPAAQPEV EKPQKKPVIK PLTEDSKKRS YNLISNDSTF
TQYRSWYLAY NYGDPQTGIR SWTLLCTPDV TCGSEQVYWS LPDMMQDPVT FRSTSQISNF PVVGAELLPV HSKSFYNDQA VYSQLIRQFT SLTHVFNRFP ENQILARPPA PTITTVSENV PALTDHGTLP LRNSIGGVQR VTITDARRRT CPYVYKALGI VSPRVLSSRT F GSGC
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Bai M, Harfe B, Freimuth P (1993) World J Gastroenterol, 67, 5198-5205 |
| Project Aim | Functional Studies |
| 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 | 37.0 |
| Expression Time | 3h |
| Expression Vector | pET11a |
| Expression Protocol | Cells were grown in LB broth at 37degC until OD600 reached 0.8. 0.4mM IPTG was added and cells were grown for a further 3h. Cells were then collected by centrifugation and washed in 10mM TrisHCl pH 8, 1mM EDTA, 100mM NaCl. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.8 |
| Cell Disruption Method | Freeze/Thaw+Sonication |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | 20mM TrisHCl pH 7.5, 1mM EDTA, 0.1% Nonidet P-40 |
| Solubilization Buffer | 6M urea |
| Refolding Buffer | 1:50mM K3PO4 pH 10.7, 50mM NaCl, 1mM EDTA, 2:PBS, 3:50mM phosphate buffer pH 7.5 |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | no tag |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Cells were lysed by 3 cycles of rapid freeze/thaw in the presence of 0.1mg/ml lysozyme, and sonicated twice for 30sec. The cell lysate was centrifuged, and the pellet was then resuspended by sonication with a cuphorn probe into wash buffer and recentrifuged. After 3 cycles of washing, the pellet was dissolved in a small amount of 6M urea, then diluted with 9 volumes of refolding buffer 1 and then dialyzed against refolding buffer 2 then refolding buffer 3. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |