Difference between revisions of "Part:BBa K5520006"
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<h2>SDS-PAGE analysis of 6×His-CsnBD78Y</h2> | <h2>SDS-PAGE analysis of 6×His-CsnBD78Y</h2> | ||
| − | Purification of the CsnB-D78Y enzyme was achieved using Ni-NTA affinity chromatography, and both the unpurified and purified proteins were confirmed via SDS-PAGE. As shown in the figure below, a distinct band at approximately 30 kDa was observed in the lane corresponding to the unpurified enzyme sample, matching the expected theoretical molecular weight. Following purification, the mutant enzyme showed a profile similar to that of CsnB, with a single band at the same molecular weight as the unpurified enzyme solution. | + | <p>Purification of the CsnB-D78Y enzyme was achieved using Ni-NTA affinity chromatography, and both the unpurified and purified proteins were confirmed via SDS-PAGE. As shown in the figure below, a distinct band at approximately 30 kDa was observed in the lane corresponding to the unpurified enzyme sample, matching the expected theoretical molecular weight. Following purification, the mutant enzyme showed a profile similar to that of CsnB, with a single band at the same molecular weight as the unpurified enzyme solution.</p> |
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Latest revision as of 04:39, 30 September 2024
6His-CsnBD78Y
This part is the rational design of the mutated gene CsnB derived from Marine Bacterium Bacilius SP. BY01 with 6×His tag.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 709
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 718
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 58
Illegal AgeI site found at 541 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
Plasmid construction
We placed 6×His tag into plasmid PET-28a for protein isolation and purification and tranformed PET-28a-6×His-CsnBD78Y into E. coli BL21 (DE3).
SDS-PAGE analysis of 6×His-CsnBD78Y
Purification of the CsnB-D78Y enzyme was achieved using Ni-NTA affinity chromatography, and both the unpurified and purified proteins were confirmed via SDS-PAGE. As shown in the figure below, a distinct band at approximately 30 kDa was observed in the lane corresponding to the unpurified enzyme sample, matching the expected theoretical molecular weight. Following purification, the mutant enzyme showed a profile similar to that of CsnB, with a single band at the same molecular weight as the unpurified enzyme solution.
