Team:Kyoto/Contribution
Contribution
This year, our team could make an innovative contribution to future iGEM, mainly through adding new parts to the iGEM registry.
First, the four parts listed below are used for peptide production. These parts are the sequences for the new purification method, ELK16 system. This system had not been adopted by other past iGEM teams. As you can see the detail of the parts from URL below, they are composed of ELK16 that has the ability to self-assemble, Mxe GyrA intein that is capable of self-disconnecting by DTT, PT linker, and the targeted protein. In this system, when the fused protein is produced, ELK16 self-aggregates and precipitates. After that, taking this aggregate and adding DTT in order to cut the N terminus of the intein, we can get the targeted protein. Compared to the other methods, the expensive column is not needed to collect peptides, and the additional process to separate the tag from the protein of interest could be omitted.
First, the four parts listed below are used for peptide production. These parts are the sequences for the new purification method, ELK16 system. This system had not been adopted by other past iGEM teams. As you can see the detail of the parts from URL below, they are composed of ELK16 that has the ability to self-assemble, Mxe GyrA intein that is capable of self-disconnecting by DTT, PT linker, and the targeted protein. In this system, when the fused protein is produced, ELK16 self-aggregates and precipitates. After that, taking this aggregate and adding DTT in order to cut the N terminus of the intein, we can get the targeted protein. Compared to the other methods, the expensive column is not needed to collect peptides, and the additional process to separate the tag from the protein of interest could be omitted.
Fig. 1
Fig. 2 BBa_K3815002
This part is to produce an antimicrobial peptide called Defensin1, which has been confirmed to be synthesized by ELK16 system.
In addition to the above parts, we also made parts composed of His tag, Mxe GyrA intein, PT linker, and the targeted protein. In the ELK16 system, the fused protein needs to be precipitated in order to recover the targeted protein[1][2][3]. Some proteins could not solubilize again once they have formed the aggregates. In this case, these parts might be useful because they have His tag instead of ELK16.This purification system with His tag and intein has contributed greatly to the development of recombinant protein production and purification because it might save time removing tag. The intein that we used this time is cut only when adding DTT. Therefore, they should be useful to improve the protein purification for future iGEM teams. In addition to this, the intein is also used for tag-free purification currently being actively researched, so these parts would be applied in a variety of ways.
In addition to the above parts, we also made parts composed of His tag, Mxe GyrA intein, PT linker, and the targeted protein. In the ELK16 system, the fused protein needs to be precipitated in order to recover the targeted protein[1][2][3]. Some proteins could not solubilize again once they have formed the aggregates. In this case, these parts might be useful because they have His tag instead of ELK16.This purification system with His tag and intein has contributed greatly to the development of recombinant protein production and purification because it might save time removing tag. The intein that we used this time is cut only when adding DTT. Therefore, they should be useful to improve the protein purification for future iGEM teams. In addition to this, the intein is also used for tag-free purification currently being actively researched, so these parts would be applied in a variety of ways.
Fig. 3 BBa_K3815007
This part is to produce an antimicrobial peptide called Defensin1, which has been confirmed to be synthesized by this tag intein method.
References
- Hai-Feng Xia, Ting-Jun Zhou, Ye-Xing Du, Yu-Jun Wang, Chang-Hua Shi, David W. Wood (2020) "Improved protein purification system based on C-terminal cleavage of Npu DnaE split intein", Bioprocess and Biosystems Engineering, 43, 1931–1941.
- Sarah F.Mitchell, Jon R.Lorsch (2015) "Chapter Eight - Protein Affinity Purification using Intein/Chitin Binding Protein Tags", Methods in Enzymology, 559, 111-125.
- Mahmoud Reza Banki, David W Wood (2005) "Inteins and affinity resin substitutes for protein purification and scale up", Microbial Cell Factories, 4, 32.