Part:BBa_K5398605
pET-28a(+)-Mfp6
The fusion part was constructed to facilitate the expression of Mfp6. The Mfp6 protein was successfully expressed in BL21(DE3) E. coli cells containing the pET28a(+) vector with the Mfp6 gene insert. After incubation at 37℃ for 5 hours, SUMO-Mfp6 (with a molecular weight of 28 kDa) was expressed, but it was found predominantly in the form of inclusion bodies. To address this, we endeavored to enhance the expression levels and employed strategies of protein denaturation and renaturation to achieve a soluble form of Mfp6.
Fig. 1 The plasmid map of pET28a(+)-Mfp6.
Fig. 2 Protein pre-expression of SUMO-Mfp6(28 kDa). Lane 1: Marker. Lane 2: Mfp6-Whole Cell Lysate(+IPTG). Lane 3: Mfp6-Supernatant(+IPTG). Lane 4: Mfp6-Pellet-PBS(+IPTG). Lane 5:Mfp6-Pellet-Extraction buffer(+IPTG) . Lane 6: Mfp6-Whole Cell Lysate-1(CK). Lane 7: Mfp6-Supernatant-1(CK). Lane 8: Mfp6-Pellet-PBS-1(CK). Lane 9:Mfp6-Pellet-Extraction buffer-1(CK) .Lane 10: Mfp6-Whole Cell Lysate-2(CK). Lane 11: Mfp6-Supernatant-2(CK). Lane 12: Mfp6-Pellet-PBS-2(CK). Lane 13:Mfp6-Pellet-Extraction buffer-2(CK).</b>
We purified SUMO-Mfp6 using a HiTrap Ni-NTA column. The purified protein was verified by SDS-PAGE and was found to be present in the XX mM imidazole elution fraction.
Fig. 3 Protein expression of SUMO-Mfp6(28 kDa).
We used a microplate reader to assay the activity of Mfp6. Fig. 4 illustrates the functionality of Mfp6. The dopamine content without Mfp6 was XX, and the dopamine content with the addition of Mfp6 was XX. These results indicate that Mfp6 significantly increases the retention of dopamine, suggesting its role in stabilizing dopamine against oxidation.
Fig. 4 Assay of Mfp6 activity.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 126
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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