Part:BBa_K2933110

GST+Linker+CPS-1

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

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

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

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

  Illegal BglII site found at 1517
  
• 23
  INCOMPATIBLE WITH RFC[23]

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

  Illegal PstI site found at 1235
  Illegal AgeI site found at 1136
  
• 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 CPS-1. It encodes a protein which is CPS-1 fused with GST tag. The fusion protein is about 59.2 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of CPS-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 CPS-1. Right: The verification results by enzyme digestion.

After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.

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