Refolding Record:
| Protein | |
|---|---|
| Protein Name | Keratoepithelin |
| Abbreviated Name | Keratoepithelin |
| SCOP Family | Unknown |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | Q15582 |
| 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 | 689 |
| Molecular Weight | 75503.7 |
| Pi | 7.66143 |
| Molecular Weight | 75503.7 |
| Disulphides | 0 |
| Full Sequence |
MALFVRLLAL ALALALGPAA TLAGPAKSPY QLVLQHSRLR GRQHGPNVCA VQKVIGTNRK YFTNCKQWYQ RKICGKSTVI SYECCPGYEK VPGEKGCPAA LPLSNLYETL GVVGSTTTQL YTDRTEKLRP EMEGPGSFTI FAPSNEAWAS LPAEVLDSLV SNVNIELLNA LRYHMVGRRV LTDELKHGMT LTSMYQNSNI QIHHYPNGIV TVNCARLLKA DHHATNGVVH LIDKVISTIT NNIQQIIEIE DTFETLRAAV AASGLNTMLE GNGQYTLLAP TNEAFEKIPS ETLNRILGDP EALRDLLNNH ILKSAMCAEA IVAGLSVETL EGTTLEVGCS GDMLTINGKA IISNKDILAT NGVIHYIDEL LIPDSAKTLF ELAAESDVST AIDLFRQAGL
GNHLSGSERL TLLAPLNSVF KDGTPPIDAH TRNLLRNHII KDQLASKYLY HGQTLETLGG KKLRVFVYRN SLCIENSCIA AHDKRGRYGT LFTMDRVLTP PMGTVMDVLK GDNRFSMLVA AIQSAGLTET LNREGVYTVF APTNEAFRAL PPRERSRLLG DAKELANILK YHIGDEILVS GGIGALVRLK SLQGDKLEVS LKNNVVSVNK EPVAEPDIMA TNGVVHVITN VLQPPANRPQ ERGDELADSA LEIFKQASAF SRASQRSVRL APVYQKLLER MKH HHHHHH
|
| Notes | Protein also has a C-terminal HSV tag, but sequence details not provided |
| Expression | |
|---|---|
| Report | Yuan C, Reuland JM, Lee L, Huang AJW (2004) Protein Expression and Purification, 35, 39-45 |
| Project Aim | Structure-Function |
| Fusion | C-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 25.0 |
| Expression Time | 4h |
| Expression Vector | pET27b |
| Expression Protocol | Cells were grown in LB medium with 0.025mg/ml kanamycin. When OD600 reached 0.6-0.8, protein expression was induced by 1mM IPTG and grown for a further 4h at 25degC, cells were harvested by centrifugation. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 600 = 0.6-0.8 |
| 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 | 6M GdnHCl, 100mM potassium phosphate, 10mM TrisHCl pH 8.0 |
| Refolding Buffer | 10mM TrisHCl, 10% glycerol, 500mM arginine hydrochloride pH 7.4 |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 7.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 0.1-1mg/ml |
| Refolding Time | 12-16hr |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Cell pellets were resuspended in BugBuster (Novagen) solution with benzonase. After incubation at room temperature for 20min with constant rocking, inclusion bodies were obtained by centrifugation (20000g, 30min, 4degC). Inclusion bodies were then resuspended in 50ml solubilization buffer and the suspension shaken overnight. Following centrifugation, the supernatant was loaded onto a pre-equilibrated column of 5ml Ni-NTA agarose. The resin was first washed with 50ml of 8M urea, 10mM potassium phosphate, 10mM TrisHCl pH 8.0, followed by 50ml of 30mM imidazole, 8M urea, 100mM potassium phosphate, 10mM TrisHCl pH 8.0. The protein was then eluted with 150mM imidazole, 8M urea, 10mM potassium phosphate, 10mM TrisHCl pH 7.4. The eluted protein samples were then mixed with 1mM DTT and incubated at room temperature for 30min. Refolding of the protein was performed using a FoldIt screening kit (Hampton Research) comprising 16 different refolding agents. Each FoldIt reagent (950microL) was mixed with 50microL of purified unfolded protein to make the final protein concentration 0.1-1mg/ml. Mixtures were incubated at 4degC with gentle rocking for 12h, then centrifuged to remove aggregates (15000g, 30min, 4degC). For large-scale refolding, the protein was diluted to 0.1mg/ml in 8M urea, 100mM potassium phosphate, 10mM TrisHCl pH 7.4, and then dialyzed at a ration of 1:250-500-fold against refolding buffer at 4degC overnight with constant stirring. |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None,L-Arginine,Glycerol |
| Additives Concentration | 10%, 0.5M |
| Refolding Yield | 70% recovery |
| Purity | |
| Notes | Various refolding buffer conditions screened using FoldIt kit (see paper for more details) |