Refolding Record:
| Protein | |
|---|---|
| Protein Name | Pepsin A |
| Abbreviated Name | Pepsin A |
| SCOP Family | Pepsin-like proteases |
| Structure Notes | |
| Organism | Pig (Sus scrofa) |
| UniProt Accession | P00791 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | n/a |
| Chimera | n/a |
| Variants | D32A |
| Chain Length | 386 |
| Molecular Weight | 41331.2 |
| Pi | 4.02653 |
| Molecular Weight | 41331.2 |
| Disulphides | 3 |
| Full Sequence |
MKWLLLLSLV VLSECLVKVP LVRKKSLRQN LIKNGKLKDF LKTHKHNPAS KYFPEAAALI GDEPLENYLD TEYFGTIGIG TPAQDFTVIF ATGSSNLWVP SVYCSSLACS DHNQFNPDDS STFEATSQEL SITYGTGSMT GILGYDTVQV GGISDTNQIF GLSETEPGSF LYYAPFDGIL GLAYPSISAS GATPVFDNLW
DQGLVSQDLF SVYLSSNDDS GSVVLLGGID SSYYTGSLNW VPVSVEGYWQ ITLDSITMDG ETIACSGGCQ AIVDTGTSLL TGPTSAIAIN IQSDIGASEN SDGEMVISCS SIDSLPDIVF TINGVQYPLS PSAYILQDDD SCTSGFEGMD VPTSSGELWI LGDVFIRQYY TVFDRANNKV GLAPVA
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Lin X-L, Wong RNS, Tang J (1989) J Biol Chem, 264, 4482-4489 |
| 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 | SG20252 |
| Expression Temp | 37.0 |
| Expression Time | not stated |
| Expression Vector | pGBT T19-99 |
| Expression Protocol | very little detail |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | French Press |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 0.1M TrisHCl pH 10, 100mM 2-mercaptoethanol |
| Solubilization Buffer | 6M urea, 100mM 2-mercaptoethanol |
| Refolding Buffer | 20mM Bis-Tris pH 6.5 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 6.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Cells were cultured for 24h at 37degC in 1L cultures of TB medium with 50mg/L ampicillin. The cells were centrifuged (3500g, 30min) and resuspended to 100ml with TN buffer (0.05M TrisHCl pH 7.2, 0.15 NaCl). 500mg lysozyme was then added and the suspension was allowed to stand at ice temperature for 30min. After freezing and thawing, 3ml of 1M MgCl2 and 1mg of DNase were added and the cells were ruptured in a French press at 2000psi. The solution was then diluted to 1L with TN buffer containing 100mM 2-mercaptoethanol and stirred overnight at 4degC and then centrifuged. The pellet was then resuspended in 1L wash buffer and stirred at 4degC overnight. The pellet was collected by centrifugation (16000g) and resuspended again in wash buffer and stirred overnight, followed by centrifugation (16000g, 30min). The pellet was then dissolved in solubilization buffer to a volume of 60ml. After stirring overnight at 4degC, the solution was centrifuged (286000g, 2h) and the supernatant was then diluted with a solution containing 6M urea, 10mM 2-mercaptoethanol, 20mM Bis-Tris pH 6.5 to a total volume of 4L. This solution was dialyzed twice against 40L of refolding buffer for 40h at 4degC. The renatured protein in was then purified further usig a Sephacryl S-300 gel filtration chromatography and FPLC anion-exchange monoQ columns. |
| Refolding Assay | Western Blot |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |