Refolding Record:
| Protein | |
|---|---|
| Protein Name | Aspartic proteinase Asp1 |
| Abbreviated Name | Asp1 |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Oryza sativa |
| UniProt Accession | ASP1_ORYSA |
| 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 | 534 |
| Molecular Weight | 58761.4 |
| Pi | 8.76 |
| Molecular Weight | 58761.4 |
| Disulphides | 0 |
| Full Sequence |
MSKQIESKTAFQEALDAAGDKLVVVDFSATWCGPCKMIKPFFHSLSEKYSNVIFLEVDVDDCQDVASECEVKCMPTFQFFKKGQKVGEFSGANKEKLEATINELVLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKPERQHMDSPVVLELHGNVYPIGHFFVTMNISDPAKPYFLDIDTGSTLTWLQCDYPCINCNKVPHGLYKPELKYAVKCTEQRCADLYADLRKPMKCGPKNQCHYGIQYVGGSSIGVLIVDSFSLPASNGTNPTSIAFGCGYNQGKNNHNVPTPVNGILGLGRGKVTLLSQLKSQGVITKHVLGHCISSKGKGFLFFGDAKVPTSGVTWSPMNREHKHYSPRQGTLHFNSNSKPISAAPMEVIFDSGATYTYFALQPYHATLSVVKSTLSKECKFLTEVKEKDRALTVCWKGKDKIRTIDEVKKCFRSLSLKFADGDKKATLEIPPEHYLIISQEGHVCLGILDGSKEHPSLAGTNLIGGITMLDQMVIYDSERSLLGWVNYQCDRIPRSASAITSRL
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Bi, X., Khush, G. S., Bennett, J. (2005) Plant Cell PHysiol, 46, 87-98 |
| Project Aim | Functional Studies,Structure-Function,Recombinant Protein Expression |
| Fusion | N-terminal hexahis + Trx Tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 30.0 |
| Expression Time | 5h |
| Expression Vector | pET32 EK.LIC |
| Expression Protocol | Cells were cultured, induced and incubated prior to extraction of fusion protein in 1x solubilization buffer(5mM imidazole, 500mM NaCl, 20mM TrisHcl, pH 7.9, 6 M Urea). |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.6 |
| Cell Disruption Method | Chemical |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 5mM imidazole, 500mM NaCl, 20mM TrisHcl, pH 7.9, 6 M Urea |
| Refolding Buffer | 50mM acetate buffer |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 3.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | overnig |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Fusion protein was purified under denaturing conditions using His Bind purification kit (Novagen). Purified protein was then refolded in 50mM acetate buffer with a pH of 3.5, concentrated with PEG6000, and dialyzed against the same buffer overnight and autolyzed in 100mM acetate buffer at room temperature for 24h. |
| Refolding Assay | Enzyme activity,Sequence Analysis |
| Refolding Chaperones | None |
| Refolding Additives | Polyethylene glycol (PEG) |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |