| Dilution |
| 20mM MOPS pH 7.8, 9M Urea |
| 20mM MOPS pH 7.8, 9M Urea |
| 20mM MOPS pH 7.8, 5mM CaCl2 |
| Ion-exchange chromatography |
| no |
| 7.8 |
| 25.0 |
| 7.5 g/L |
|
| None |
| n/a |
| Cells were grown in LB media with 2g/L glucose at 30degC and pH 6.0. After 6h of incubation, 0.5mM IPTG and 5mM CaCl2 were added to induce protein expression. Cells were harvested and resuspended in 50mM sodium phosphate buffer (pH 6.0). Cells were lysed using a French press at 30000psi and centrifuged. The supernatant was applied to an alpha-cyclodextrin-coupled sepharose 6B column. The column was washed with sodium phosphate, then the protein was eluted with sodium phosphate buffer containing 1%(w/v) beta-cyclodextrin. The eluted protein was then dialyzed against 20mM MOPS pH 7.0 for 48 hours with intermittent buffer changes.
The protein was then denatured by incubation in solubilization buffer for more than 16 hours. The protein was then loaded onto an SP-sepharose resin equilibrated in solubilization buffer. The protein and resin were mixed gently for 2h, then unbound proteins were removed by washing with solubilization buffer. Refolding of the immobilized protein was induced by addition of 19 volumes of refolding buffer to the resin. |
| Enzyme activity |
| None |
| None |
| NULL |
| nearly 100% |
|
| Protein expressed and purified with and without C-terminal (Lys10) tag - polycationic fusion assists immobilization onto SP sepharose.
Effects of variation in NaCl concentration, pH, protein concentration on refolding yields also investigated - see paper for more details
Protein expressed and purified in native form, then denatured and refolded
Sequence:
MPD TSVDNKVNFS TDVIYQIVTD 50
RFADGDRTNN PAGDAFSGDR SNLKLYFGGD WQGIIDKIND GYLTGMGVTA 100
LWISQPVENI TSVIKYSGVN NTSYHGYWAR DFKQTNDAFG DFADFQNLID 150
TAHAHNIKVV IDFAPNHTSP ADRDNPGFAE NGGMYDNGSL LGAYSNDTAG 200
LFHHNGGTDF STIEDGIYKN LYDLADINHN NNAMDAYFKS AIDLWLGMGV 250
DGIRFDAVKH MPFGWQKSFV SSIYGGDHPV FTFGEWYLGA DQTDGDNIKF 300
ANESGMNLLD FEYAQEVREV FRDKTETMKD LYEVLASTES QYDYINNMVT 350
FIDNHDMDRF QVAGSGTRAT EQALALTLTS RGVPAIYYGT EQYMTGDGDP 400
NNRAMMTSFN TGTTAYKVIQ ALAPLRKSNP AIAYGTTTER WVNNDVLIIE 450
RKFGSSAALV AINRNSSAAY PISGLLSSLP AGTYSDVLNG LLNGNSITVG 500
SGGAVTNFTL AAGGTAVWQY TAPETSPAIG NVGPTMGQPG NIVTIDGRGF 550
GGTAGTVYFG TTAVTGSGIV SWEDTQIKAV IPKVAAGKTG VSVKTSSGTA 600
SNTFKSFNVL TGDQVTVRFL VNQANTNYGT NVYLVGNAAE LGSWDPNKAI 650
GPMYNQVIAK YPSWYYDVSV PAGTKLDFKF IKKGGGTVTW EGGGNHTYTT 700
PASGVGTVTV DWQN KKKKKKKKKK
Number of amino acids: 697
Molecular weight: 75338.6
Theoretical pI: 5.24
|