Refolding Record:
| Protein | |
|---|---|
| Protein Name | Pertactin Cleaved into: Outer membrane protein P.69 |
| Abbreviated Name | P.69 Pertactin |
| SCOP Family | Virulence factor P.69 pertactin |
| Structure Notes | |
| Organism | Bordetella pertussis |
| UniProt Accession | P14283 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 678 |
| Molecular Weight | 68642.7 |
| Pi | 6.65 |
| Molecular Weight | 68642.7 |
| Disulphides | Unknown |
| Full Sequence |
DWNNQSIVKTGERQHGIHIQGSDPGGVRTASGTTIKVSGRQAQGILLENPAAELQFRNGSVTSSGQLSDDGIRRFLGTVTVKAGKLVADHATLANVGDTWDDDGIALYVAGEQAQASIADSTLQGAGGVQIERGANVTVQRSAIVDGGLHIGALQSLQPEDLPPSRVVLRDTNVTAVPASGAPAAVSVLGASELTLDGGHITGGRAAGVAAMQGAVVHLQRATIRRGDAPAGGAVPGGAVPGGAVPGGFGPGGFGPVLDGWYGVDVSGSSVELAQSIVEAPELGAAIRVGRGARVTVSGGSLSAPHGNVIETGGARRFAPQAAPLSITLQAGAHAQGKALLYRVLPEPVKLTLTGGADAQGDIVATELPSIPGTSIGPLDVALASQARWTGATRAVDSLSIDNATWVMTDNSNVGALRLASDGSVDFQQPAEAGRFKVLTVNTLAGSGLFRMNVFADLGLSDKLVVMQDASGQHRLWVRNSGSEPASANTLLLVQTPLGSAATFTLANKDGKVDIGTYRYRLAANGNGQWSLVGAKAPPAPKPAPQPGPQPPQPPQPQPEAPAPQPPAGRELSAAANAAVNTGGVGLASTLWYAESNALSKRLGELRLNPDAGGAWGRGFAQRQQLDNRAGRRFDQKVAGFELGADHAVAVAGGRWHLGGLAGYTRGDRGFTGDGGG
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Hijnen M, van Gageldonk PG, Berbers GA, van Woerkom T, Mooi FR. (2005) Protein Expression and Purification, 41, 106-112 |
| Project Aim | Vaccine 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 | 4 h |
| Expression Vector | pET |
| Expression Protocol | This strain partially compensates possible codon bias when expressing high GC-content genes in E. coli. A single colony of a transformant was used to inoculate 5 ml LB media containing 100 μg/ml ampicillin and 50 μg/ml chloramphenicol. Bacteria were grown at 37 °C overnight at 250 rpm. From the overnight cultures, 100 μl was added to 1 L fresh LB media containing 100 μg/ml ampicillin and 50 μg/ml chloramphenicol. Cultures were incubated at 37 °C at 250 rpm until the OD600 reached 0.6–0.8. Subsequently, cultures were induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG), and incubated further for 4 h. Bacteria were harvested by centrifugation at 5000g for 10 min at 4 °C. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.6-0.8 = 600 |
| Cell Disruption Method | Not stated |
| Lytic Agent | Lysozyme |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | Bug Buster reagent |
| Solubilization Buffer | 6 M guanidine hydrochloride (GuHCl), 10 mM benzamidine, 1 mM EDTA, 100 mM NaCl, and 50 mM Tris/HCl, pH 8.8 |
| Refolding Buffer | 10 mM benzamidine, 1 mM EDTA, 100 mM NaCl, and 50 mM Tris/HCl, pH 8.8 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.8 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | overnight |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | nclusion bodies were isolated using the Bug Buster protein extraction reagent (Novagen, Darmstadt, Germany) according to the protocol provided by the manufacturer. Briefly, induced cells were harvested and subsequently lysed using Bug Buster reagent. The cell lysate was treated with 5000 U lysozyme and 125 U benzonase nuclease per gram of wet cell paste. Inclusion bodies were collected by centrifugation and were washed three times with 1:10 diluted Bug Buster reagent. The purified inclusion bodies were solubilized in 6 M guanidine hydrochloride (GuHCl), 10 mM benzamidine, 1 mM EDTA, 100 mM NaCl, and 50 mM Tris/HCl, pH 8.8. Refolding of the recombinant pertactin and variants was initiated by rapid 50-fold dilution into the same buffer without GuHCl. Proteins were allowed to fully refold during overnight dialysis at 4 °C against 1 mM EDTA, 100 mM NaCl, and 50 mM Tris/HCl, pH 8.8. Subsequently, the refolded proteins were dialyzed twice against 50 mM Tris/HCl, pH 8.8, using Spectra/Por 7 dialysis membranes with a molecular weight cut-off (MWCO) of 50 kDa (Spectrum Laboratories, Rancho Dominguez, CA). The proteins were concentrated on an Amicon Ultra-15 concentrator with a 50 kDa cut-off (Millipore, Billerica, MA). Finally, 2 μg protease inhibitor (Roche, Penzberg, Germany) was added to 1 mg/ml of the concentrated proteins. |
| Refolding Assay | Western Blot,SDS-PAGE,Circular Dichroism (uv-CD) |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |