Part:BBa_K5520006

6�His-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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 709
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 718
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 58
  Illegal AgeI site found at 541
  
• 1000
  COMPATIBLE WITH RFC[1000]

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.