| Curtis-Fisk J, Spencer RM, Weliky DP.
(2008)
Protein Expression and Purification,
1,
1 |
| Protein refolding |
| C-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| E coli |
| BL21(DE3) |
| 37.0 |
| 3 h |
| n/a |
| All cell cultures were grown in media containing 15 mg/L kanamycin. Bacterial growth was initiated from a glycerol stock of the recombinant bacterial cells to 1 L of “enriched LB” which contained LB supplemented with 10 mL glycerol. The cell suspension was grown overnight to maximum cell density in a 2.8 L baffled Fernbach flask with a foam closure, shaking at 37 °C and 140 rpm. The cell suspension was then centrifuged at 10,000g for 10 min to produce a solid cell pellet. The pellet was resuspended into 1 L of minimal medium whose optimal composition included the commercial M9 minimal medium salts (6.8 g/L Na2HPO4, 3.0 g/L NaH2PO4, 0.50 g/L NaCl, 1.0 g/L NH4Cl), 2.5 g/L MgSO4, and 10 g/L glycerol at pH 8.0. Cell growth was continued by shaking at 37 °C at 140 rpm.
Many of the experiments in this paper examine the effect of a parameter such as glucose concentration on cell growth. Each growth optimization experiment was done using a 100 mL culture volume in a 250 mL baffled Erlenmeyer flask and all of the growths as a function of a single parameter were performed at the same time in the same shaker. All of the comparative experiments presented in this paper were repeated and for each parameter, the trends in cell growth were reproducible. The value of the parameter which yielded highest growth was also reproducible. Examination of all of the A600 or ΔA600 values between the different comparative growths showed that there was systematic variation by as much as 20%, e.g. for each parameter value, (A600)first growth/(A600)second growth would be 1.2. It was therefore difficult to estimate error bars which would reflect data uncertainties within a single comparative growth.Isotopically labeled FHA2 production. After 1 h of resuspended cell growth in optimized minimal medium, FHA2 expression was induced by addition of isopropyl thiogalactoside (IPTG) to a final concentration of 0.2 mM. For production of FHA2 with 1-13C, 15N Leu isotopic labeling, 100 mg/L of labeled amino acid was added at the time of protein induction. FHA2 production was continued for 3 h at 37 °C. The cell pellet was harvested by centrifugation at 10,000g for 10 min, and the pellet was then stored at −80 °C until purification. |
| IPTG |
| OD 1.2 =
600 |
| Sonication |
| None |
| Metal affinity chromatography |
| partial |
| Dilution |
| 50 mM sodium phosphate at pH 8.0, 300 mM NaCl, 20 mM imidazole, and 0.5% (w/v) N-lauroylsarcosine (Sarkosyl) |
| 8 M urea, 100 mM NaH2PO4, 10 mM Tris–Cl |
| 1 M arginine, 10 mM Tris–Cl, 0.17% decyl maltoside, 2 mM EDTA at pH 8 |
| Metal affinity chromatography |
| yes |
| 8.0 |
| 4.0 |
| n/a |
| overnight |
| None |
| n/a |
| The inclusion body fraction of FHA2 was defined as the component which pelleted during the centrifugation of the cell lysate. The pellet was sonicated in denaturing lysis buffer (8 M urea, 100 mM NaH2PO4, 10 mM Tris–Cl), the suspension centrifuged, and the supernatant purified with chelated cobalt His-Select resin using methods similar to those described above. After binding the denatured protein to the resin, the column was washed with 3 column volumes of denaturing wash buffer (8 M urea, 100 mM NaH2PO4, 10 mM Tris–Cl, 20 mM imidazole) and the FHA2 was eluted with 5 column volumes of denaturing elution buffer (8 M urea, 100 mM NaH2PO4, 10 mM Tris–Cl, 250 mM imidazole). The denatured FHA2 solution was rapidly diluted into twice the volume of ice cold refolding buffer (1 M arginine, 10 mM Tris–Cl, 0.17% decyl maltoside, 2 mM EDTA at pH 8) and stored at 4 °C overnight. Removal of urea and refolding of FHA2 was achieved with dialysis at 4 °C for two days using 10,000 MWCO tubing and a dialysis buffer [7]. |
| NMR analysis,Circular Dichroism (uv-CD) |
| None |
| L-Arginine |
| 1 M |
| 10 mg/L |
| n/a |
| n/a |