Difference between revisions of "Part:BBa K2933139"
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<partinfo>BBa_K2933139 parameters</partinfo> | <partinfo>BBa_K2933139 parameters</partinfo> | ||
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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 IMP-71. It encodes a protein which is IMP-71 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 IMP-71 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]Purification, crystallization and preliminary X-ray analysis of IMP-18, a class B carbapenemase from Pseudomonas aeruginosa.Furuyama T, Ishii Y, Ohya N, Tateda K, Hanson ND, Shimizu-Ibuka A.Acta Crystallogr Sect F Struct Biol Cryst Commun. 2013 Dec;69(Pt 12):1397-400. | ||
+ | |||
+ | ===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.<br> | ||
+ | <p style="text-align: center;"> | ||
+ | [[File:IMP-71-PCR.png|400px]]<br> | ||
+ | '''Figure 1.''' Left: The PCR result of IMP-71. Right: The verification results by enzyme digestion.<br> | ||
+ | </p> | ||
+ | After verification, it was determined that the construction is successful. We converted the plasmid to ''E. coli'' BL21(DE3) for expression and purification.<br> |
Revision as of 14:02, 24 September 2019
His+Linker a+Sumo+Linker b+IMP-71
This part encodes the fusion protein of His tag, sumo tag and IMP-71 to promote the expression and purification of target protein(IMP-71).
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 256
Illegal PstI site found at 1079 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 256
Illegal NheI site found at 33
Illegal PstI site found at 1079 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 256
Illegal BglII site found at 145
Illegal BamHI site found at 344 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 256
Illegal PstI site found at 1079 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 256
Illegal PstI site found at 1079 - 1000COMPATIBLE WITH RFC[1000]
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 IMP-71. It encodes a protein which is IMP-71 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 IMP-71 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]Purification, crystallization and preliminary X-ray analysis of IMP-18, a class B carbapenemase from Pseudomonas aeruginosa.Furuyama T, Ishii Y, Ohya N, Tateda K, Hanson ND, Shimizu-Ibuka A.Acta Crystallogr Sect F Struct Biol Cryst Commun. 2013 Dec;69(Pt 12):1397-400.
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 IMP-71. 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.