Refolding Record:
| Protein | |
|---|---|
| Protein Name | Penicillin-Binding Protein 2a |
| Abbreviated Name | PBP |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Streptococcus pneumoniae |
| UniProt Accession | P0A3M5 |
| 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 | 691 |
| Molecular Weight | 73872.9 |
| Pi | 5.10255 |
| Molecular Weight | 73872.9 |
| Disulphides | Unknown |
| Full Sequence |
MRKFNSHSIPIRLNLLFSIVILLFMTIIGRLLYMQVLNKDFYEKKLASASQTKITSSSAR
GEIYDASGKPLVENTLKQVVSFTRSNKMTATDLKETAKKLLTYVSISSPNLTERQLADYY
LADPEIYKKIVEALPSEKRLDSDGNRLSESELYNNAVDSVQTSQLNYTEDEKKEIYLFSQ
LNAVGNFATGTIATDPLNDSQVAVIASISKEMPGISISTSWDRKVLETSLSSIVGSVSSE
KAGLPAEEAEAYLKKGYSLNDRVGTSYLEKQYEETLQGKRSVKEIHLDKYGNMESVDTIE
EGSKGNNIKLTIDLAFQDSVDALLKSYFNSELENGGAKYSEGVYAVALNPKTGAVLSMSG
IKHDLKTGELTPDSLGTVTNVFVPGSVVKAATISSGWENGVLSGNQTLTDQSIVFQGSAP
INSWYTQAYGSFPITAVQALEYSSNTYMVQTALGLMGQTYQPNMFVGTSNLESAMEKLRS
TFGEYGLGTATGIDLPDESTGFVPKEYSFANYITNAFGQFDNYTPMQLAQYVATIANNGV
RVAPRIVEGIYGNNDKGGLGDLIQQLQPTEMNKVNISDSDMSILHQGFYQVAHGTSGLTT
GRAFSNGALVSISGKTGTAESYVADGQQATNTNAVAYAPSDNPQIAVAVVFPHNTNLTNG
VGPSIARDIINLYQKYHPMN
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Zhao G, Meier TI, Hoskins J, Jaskunas SR. (1999) Protein Expression and Purification, 16, 331-339 |
| Project Aim | Structural Studies |
| Fusion | N-terminal GST |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | DH5alpha |
| Expression Temp | 32.0 |
| Expression Time | 3h |
| Expression Vector | pGEX-2T |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 50mM Tris-HCl, 1mM EDTA, 100mM KCl, pH 7.5 |
| Solubilization Buffer | 7.5M urea |
| Refolding Buffer | 50mM Tris-HCl, 1mM EDTA, 100mM KCl |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | 16h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The inclusion bodies were washed 6 times with 50mM Tris-HCl, 1mM EDTA, 100mM KCl, pH 7.5. The protein was solubilixed with 7.5M urea and refolded by dialysis against 50mM Tris-HCl, 1mM EDTA, 100mM KCl, pH 7.5. |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 37mg/L culture - 36% |
| Purity | 90-95% pure |
| Notes | |