Refolding Record:
| Protein | |
|---|---|
| Protein Name | Penicillin-Binding Protein 2a |
| Abbreviated Name | PBP |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Staphylococcus aureus |
| UniProt Accession | P0A3M5 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Unknown |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | N-terminal truncated form (misisng aa.1-22) |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 646 |
| Molecular Weight | 73630.3 |
| Pi | 8.42225 |
| Molecular Weight | 73630.3 |
| Disulphides | 0 |
| Full Sequence |
YASKDKEI NNTIDAIEDK NFKQVYKDSS YISKSDNGEV EMTERPIKIY NSLGVKDINI QDRKIKKVSK NKKRVDAQYK
IKTNYGNIDR NVQFNFVKED GMWKLDWDHS VIIPGMQKDQ SIHIENLKSE RGKILDRNNV ELANTGTAYE IGIVPKNVSK KDYKAIAKEL SISEDYIKQQ MDQNWVQDDT FVPLKTVKKM DEYLSDFAKK FHLTTNETES RNYPLGKATS HLLGYVGPIN SEELKQKEYK GYKDDAVIGK KGLEKLYDKK LQHEDGYRVT
IVDDNSNTIA HTLIEKKKKD GKDIQLTIDA KVQKSIYNNM KNDYGSGTAI HPQTGELLAL VSTPSYDVYP FMYGMSNEEY NKLTEDKKEP LLNKFQITTS PGSTQKILTA MIGLNNKTLD DKTSYKIDGK GWQKDKSWGG YNVTRYEVVN GNIDLKQAIE SSDNIFFARV ALELGSKKFE KGMKKLGVGE DIPSDYPFYN
AQISNKNLDN EILLADSGYG QGEILINPVQ ILSIYSALEN NGNINAPHLL KDTKNKVWKK NIISKENINL LTDGMQQVVN KTHKEDIYRS YANLIGKSGT AELKMKQGET GRQIGWFISY DKDNPNMMMA INVKDVQDKG MASYNAKISG KVYDELYENG NKKYDIDE
|
| Notes | NB: potential active site mutants S403C or S403A also expressed and purified. |
| Expression | |
|---|---|
| Report | Frank LJ, Wisniewski D, Hammond GG, Hermes J, Marcy A, Cameron PM (1995) Protein Expression and Purification, 6, 671-678 |
| Project Aim | Drug 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 | 2h |
| Expression Vector | pET11d |
| Expression Protocol | 6x1L batches of M9/Amp media were inoculated with 35ml overnight stock culture. Flasks were incubated with shaking at 37degC until A600 reached 0.4. 0.1mM IPTG was added and incubation continued for a further 2h. Cells were harvested by centrifugation (4000g, 10min). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.4 |
| Cell Disruption Method | French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | 1: PBS, 25% sucrose, 5mM EDTA, 1% Triton X-100. 2:1M urea, 10mM Tris pH 7.0 |
| Solubilization Buffer | 5M GdnHCl, 50mM Tris pH 8.0, 0.5M NaCl, 0.01% thiodiglycol |
| Refolding Buffer | 50mM sodium bicarbonate pH 8.0, 500mM NaCl, 20% glycerol, 0.01% thiodiglycol |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 5.0 |
| Protein Concentration | 50microg/ml |
| Refolding Time | 4h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | A total of 8.8g wet cell paste was resuspended in 75ml of 100mM Tris pH 8.0 and frozen at -70degC. The cells were thawed at room temperature and a protease inhibitor cocktail was added (1microg/ml each of bestatin, leupeptin, aprotinin and pepstatin). Cells werer lysed by two passes through a French press (20000psi, 5degC) and 20000U of DNasI was added, followed by CaCl2 and MgCl2 to 1mM each. The solution was mixed for 30min at room temp, followed by centrifugation (37000g, 15min). The pellet was washed once with wash buffer 1, followed by 3 washes with wash buffer 2. The inclusion body pellets were dissolved in 15ml solubilization buffer. The solution was briefly sonicated in a water bath, followed by rocking at room temperature for 50min. The mixture was then centrifuged (30min, 37000g) and the supernatant was added to 500ml refolding buffer. The solution was stirred gently at 5degC for 4h, followed by centrifugation (37000g, 30min). The supernatant was dialyzed against 20L of cold 50mM Na bicarbonate pH 8.0, 0.01% thiodiglycol and three successivly lower concentrations of NaCl. Over the course of two days, the concentration of NaCl was decreased step-wise from 0.5 to 0.15M to a final concentration of 5mM. The dialysate was centrifuged (10000g, 30min) and filtered. The protein was then purified further by anion exchange chromatography and size exclusion chromatography. |
| Refolding Assay | HPLC |
| Refolding Chaperones | None |
| Refolding Additives | None,Glycerol |
| Additives Concentration | 20% |
| Refolding Yield | 43% |
| Purity | >98% |
| Notes | |