Part:BBa_K2933106
GST+Linker+ElBlaII
This part encodes the fusion protein of GST tag and ElBla2-1 to promote the expression and purification of target protein(ElBla2-1).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1182
- 1000INCOMPATIBLE 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 ElBla2-1. It encodes a protein which is ElBla2-1 fused with GST tag. The fusion protein is about 53.5 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of ElBla2-1 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 Elbla2-1. 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.
Expression and purification
Pre-expression:
The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.
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