fusion protein to increase the amount of Z-ISO
containing heme.
3. Repeat multiple rounds of cell cultures to combine for later
processing if large quantities of purified protein are desired.
4. From this point on, keep all samples and solutions on ice.
5. Choose a beaker size such that the volume of the sample is
about 1/3–1/4 of the beaker and the sonication probe remans
immerged in the sample fluid. Sonications should be done in an
ice bucket filled with ice and water so that the sample stays cold.
To prevent hearing loss, place the ice bucket containing the
protein sample in a closed hood and sonicate with ear protectors or ear plugs. After sonication, as the ice melts from the
heat, add more ice and remove some water to maintain cold
temperature for the sample. The number of sonications
depends on the color observed after sonication. The supernatant should go from a milky pink to a deep translucent red (see
Fig. 2a). If the sample is insufficiently sonicated, the protein
yield is reduced. If oversonicated, protein quality is affected.
The sample can be divided into multiple batches, if needed.
6. This step is important for purifying the membrane fraction
containing MBP::Z-ISO. Carefully remove the supernatant
where there is likely an abundance of a truncated MBP product
but not the MBP::Z-ISO fusion protein.
7. When adding buffers to the resin, make sure that the entire
resin is mixed with buffer.
8. Do not elute the His-tagged Z-ISO with imidazole as imidazole binds to the heme.
9. Protein samples can be concentrated using centrifugal microconcentrators from vendors such as Amicon.
10. Depending on the subsequent analyses, the sample can be
further concentrated after dialysis. For electron paramagnetic
resonance (EPR) studies, use ~5 mg protein samples at a
concentration of ~15.1 mg mL
À1 (~200 μM); for magnetic
circular dichroism (MCD), use 1.6 mg protein samples at
1.57 mg mL
À1 (~20 μM); for inductively coupled plasma
optical emission spectrometry (ICP-OES), use 1 mg at
1 mg mL
À1 [5].
Acknowledgments
Dr. AImin Liu (The University of Texas at San Antonio) provided
helpful advice on optimizing heme biosynthesis for maximal hemeloading of Z-ISO produced in E. coli. Research in the Wurtzel
laboratory has been funded by the National Institutes of Health
60
Eleanore T. Wurtzel and Jesu ´ s Beltra ´ n
containing heme.
3. Repeat multiple rounds of cell cultures to combine for later
processing if large quantities of purified protein are desired.
4. From this point on, keep all samples and solutions on ice.
5. Choose a beaker size such that the volume of the sample is
about 1/3–1/4 of the beaker and the sonication probe remans
immerged in the sample fluid. Sonications should be done in an
ice bucket filled with ice and water so that the sample stays cold.
To prevent hearing loss, place the ice bucket containing the
protein sample in a closed hood and sonicate with ear protectors or ear plugs. After sonication, as the ice melts from the
heat, add more ice and remove some water to maintain cold
temperature for the sample. The number of sonications
depends on the color observed after sonication. The supernatant should go from a milky pink to a deep translucent red (see
Fig. 2a). If the sample is insufficiently sonicated, the protein
yield is reduced. If oversonicated, protein quality is affected.
The sample can be divided into multiple batches, if needed.
6. This step is important for purifying the membrane fraction
containing MBP::Z-ISO. Carefully remove the supernatant
where there is likely an abundance of a truncated MBP product
but not the MBP::Z-ISO fusion protein.
7. When adding buffers to the resin, make sure that the entire
resin is mixed with buffer.
8. Do not elute the His-tagged Z-ISO with imidazole as imidazole binds to the heme.
9. Protein samples can be concentrated using centrifugal microconcentrators from vendors such as Amicon.
10. Depending on the subsequent analyses, the sample can be
further concentrated after dialysis. For electron paramagnetic
resonance (EPR) studies, use ~5 mg protein samples at a
concentration of ~15.1 mg mL
À1 (~200 μM); for magnetic
circular dichroism (MCD), use 1.6 mg protein samples at
1.57 mg mL
À1 (~20 μM); for inductively coupled plasma
optical emission spectrometry (ICP-OES), use 1 mg at
1 mg mL
À1 [5].
Acknowledgments
Dr. AImin Liu (The University of Texas at San Antonio) provided
helpful advice on optimizing heme biosynthesis for maximal hemeloading of Z-ISO produced in E. coli. Research in the Wurtzel
laboratory has been funded by the National Institutes of Health
60
Eleanore T. Wurtzel and Jesu ´ s Beltra ´ n