| Eisenmesser EZ, Kapust RB, Nawrocki JP, Mazzulla MJ, Pannell LK, Waugh DS, Byrd RA
(2000)
Protein Expression and Purification,
20,
186-195 |
| Structure-Function |
| N-terminal maltose binding protein |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21 |
| 37.0 |
| 24h |
| pMAL-c2 |
| For a typical 2-L preparation, an aliquot was taken from the 10-ml culture
grown in LB broth and added to a 100-ml volume of M9 medium to an OD600 of 0.1. The M9 medium was modified to contain a total of 3 g per liter of glucose and
100 mg/ml ampicillin. The 100-ml culture was grown at 37degC until OD600 reached 2.0 (approximately 8–12 h). The culture was diluted into 2 L of M9 medium, yielding an initial OD600 of approximately 0.1. The cells were grown to an OD600 of 0.3, at which time IPTG was added to a final concentration of 1.0 mM. After 24 h at 37degC, the cells were harvested by centrifugation, yielding approximately 3 g of wet cell paste per liter of
culture and frozen at -80degC.
|
| IPTG |
| OD 600 =
0.3 |
| Sonication |
| None |
| Size-exclusion chromatography |
| insoluble |
| Dilution/Dialysis combination |
| 50mM Tris-HCl, 200mM NaCl, 1mM EDTA, 2M urea, pH 7.6 |
| 100mM Na2HPO4, 100mM DTT, 8M guanidinium chloride pH 6.0 |
| 50mM Tris-HCl, 50mM NaCl, 1M glycine, 1mM EDTA, 5mM beta mercaptoethanol |
| Size-exclusion chromatography |
| yes |
| 8.0 |
| 25.0 |
|
| 5h |
| Beta-mercaptoethanol |
| 5mM |
| The IL13-MBP fusion was coexpressed in E.coli with TEV protease (pRK603) allowing for expression and intracellular cleavage of the target protein which aggregated into inclusion bodies. Inclusion bodies were purified under harsh conditions (using rounds of sonication in wash buffer) which resulted in enhanced final purity. The inclusion bodies were solubilised in solubilisation buffer and pre purified by gel filtration (Sephacryl S-200) using a running buffer of 50mM Na2HPO4, 1M NaCl, 1mM EDTA, 20mM beta mercaptoethanol, 4M guanidinium chloride. Peaks containing IL-13 were refolded by a 50-fold drop-wise dilution into refolding buffer and subsequently dialysed against 25mM Na2HPO4, 10mM NaCl, 1mM EDTA pH 6.1. |
| 15N -1H chemical shifts (ppm) |
| None |
| None,Glycine |
| 1M |
| 4mg/1L media |
|
|