Part:BBa_K2933109

GST+Linker+PEDO-1

This part encodes the fusion protein of GST tag and PEDO-1 to promote the expression and purification of target protein(PEDO-1).

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1235
  Illegal PstI site found at 1521
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1235
  Illegal PstI site found at 1521
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1235
  Illegal PstI site found at 1521
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1235
  Illegal PstI site found at 1521
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 85
  

Usage and Biology

This composite part is made up with three basic parts, the GST tag, the cutting site of Prescission Protease and our target protein PEDO-1. It encodes a protein which is PEDO-1 fused with GST tag. The fusion protein is about 58.2 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of PEDO-1 and combine the two parts with the cutting site of Prescission Protease. The fusion protein can be cut off at the cutting site by Prescission Protease. It is convenient for us to purify our target protein.

Molecular cloning

First, we used the vector pGEX-6p-1 to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.

Figure 1. Left: The PCR result of PEDO-1. Right: The verification results by enzyme digestion.

References

[1]Dereje Dadi Gudeta, Valeria Bortolaia,The Soil Microbiota Harbors a Diversity of Carbapenem-Hydrolyzing β-Lactamases of Potential Clinical Relevance[J]，Antimicrobial Agents and chemotherapy,January 2016