Difference between revisions of "Part:BBa K2933134"

Revision as of 11:41, 25 September 2019

His+Linker a+Sumo+Linker b+TMB-2

This part encodes the fusion protein of His tag, sumo tag and TMB-2 to promote the expression and purification of target protein(TMB-2).

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 256
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 256
Illegal NheI site found at 33
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 256
Illegal BglII site found at 145
Illegal BamHI site found at 344
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 256
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 256
1000
COMPATIBLE WITH RFC[1000]

Usage and Biology

This composite part is made up with five basic parts, the His tag, the Sumo tag, three cutting sites(NdeI, NheI and BamHI) and our target protein TMB-2. It encodes a protein which is TMB-2 fused with His-Sumo tag. The fusion protein is about 39.2kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of TMB-2 and combine the three parts with these three cutting sites. The fusion protein can be cut off at the cutting site BamHI. It is convenient for us to purify our target protein.

References

[1]Structural Insights into TMB-1 and the Role of Residues 119 and 228 in Substrate and Inhibitor Binding.Skagseth S, Christopeit T, Akhter S, Bayer A, Samuelsen Ø, Leiros HS.Antimicrob Agents Chemother. 2017 Jul 25;61(8).

Molecular cloning

First, we used the vector pGEX-6p-1 and pET-28b_SUMO 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 TMB-2. 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.

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 ===Usage and Biology===
-This composite part is made up with four basic parts, the His tag, the Sumo tag, three cutting sites(NdeI, NheI and BamHI) and our target protein TMB-2. It encodes a protein which is TMB-2 fused with His-Sumo tag. The fusion protein is about 39.2kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of TMB-2 and combine the three parts with these three cutting sites. The fusion protein can be cut off at the cutting site BamHI. It is convenient for us to purify our target protein.<br>
+This composite part is made up with five basic parts, the His tag, the Sumo tag, three cutting sites(NdeI, NheI and BamHI) and our target protein TMB-2. It encodes a protein which is TMB-2 fused with His-Sumo tag. The fusion protein is about 39.2kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of TMB-2 and combine the three parts with these three cutting sites. The fusion protein can be cut off at the cutting site BamHI. It is convenient for us to purify our target protein.<br>
 ===References===
 [1]Structural Insights into TMB-1 and the Role of Residues 119 and 228 in Substrate and Inhibitor Binding.Skagseth S, Christopeit T, Akhter S, Bayer A, Samuelsen Ø, Leiros HS.Antimicrob Agents Chemother. 2017 Jul 25;61(8).