Difference between revisions of "Part:BBa K5520009"
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Revision as of 05:38, 30 September 2024
6*His-CsnBK260Y
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-CsnBK260Y into E. coli BL21 (DE3).
SDS-PAGE analysis of 6×His-CsnBV186Y
The purification of the CsnB-K260Y enzyme was accomplished using Ni-NTA affinity chromatography, which facilitated the isolation of the target protein due to the presence of a 6×His tag. The effectiveness of the purification process was confirmed by SDS-PAGE analysis of both the unpurified and purified protein samples. As depicted in the figure below, a specific lane 2 corresponding to the mutant enzyme was observed at approximately 30 kDa in the unpurified sample, consistent with the expected molecular weight. After purification, the mutant enzyme demonstrated a profile reminiscent of the wild-type CsnB, displaying a single band at the same molecular weight as the unpurified enzyme solution.