Difference between revisions of "Part:BBa K4241023"
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This is a bacterial expression system for SUMO-RFP fusion protein. By expressing the T7 tagged SUMO-RFP fusion protein via an enhanced T7pCONS system, we are able to increase the expression yield of the protein. The nature of the SUMO-tag further allows for a simplified purification scheme. | This is a bacterial expression system for SUMO-RFP fusion protein. By expressing the T7 tagged SUMO-RFP fusion protein via an enhanced T7pCONS system, we are able to increase the expression yield of the protein. The nature of the SUMO-tag further allows for a simplified purification scheme. | ||
<br/> | <br/> | ||
| − | Purification scheme: | + | Purification scheme: <br/> |
| − | 1. Express the fusion protein in E. coli and sonicate cells | + | 1. Express the fusion protein in E. coli and sonicate cells <br/> |
| − | 2. His-pulldown the cell lysate | + | 2. His-pulldown the cell lysate <br/> |
| − | 3. Cleave with Ulp1 | + | 3. Cleave with Ulp1 <br/> |
4. His-pulldown to seperate uncleaved proteins and non-target proteins. | 4. His-pulldown to seperate uncleaved proteins and non-target proteins. | ||
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===Parts that comprise this composite component=== | ===Parts that comprise this composite component=== | ||
| − | Enhance-T7pro-RBS-Start-6xHis-T7tag - Part:BBa_K4241016 | + | Enhance-T7pro-RBS-Start-6xHis-T7tag - Part:BBa_K4241016 <br/> |
| − | SUMO (Yeast with mutation) - Part:BBa_K4241022 | + | SUMO (Yeast with mutation) - Part:BBa_K4241022 <br/> |
| − | highly engineered mutant of red fluorescent protein from Discosoma striata (coral) - Part:BBa_E1010 | + | highly engineered mutant of red fluorescent protein from Discosoma striata (coral) - Part:BBa_E1010 <br/> |
| − | Double stop codon - Part:BBa_K4241017 | + | Double stop codon - Part:BBa_K4241017 <br/> |
| − | double terminator (B0010-B0012) - Part:BBa_B0015 | + | double terminator (B0010-B0012) - Part:BBa_B0015 <br/> |
Latest revision as of 16:26, 9 October 2022
6xHis_SUMO_RFP
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 155
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 301
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1050
Illegal AgeI site found at 1162 - 1000COMPATIBLE WITH RFC[1000]
Overview
This composite part is for the expression of a T7 tagged, SUMO-RFP fusion protein. This allows for the production and purification of the RFP via a double His-tag purification scheme by cleaving SUMO with Ulp1.
Usage and Biology
This is a bacterial expression system for SUMO-RFP fusion protein. By expressing the T7 tagged SUMO-RFP fusion protein via an enhanced T7pCONS system, we are able to increase the expression yield of the protein. The nature of the SUMO-tag further allows for a simplified purification scheme.
Purification scheme:
1. Express the fusion protein in E. coli and sonicate cells
2. His-pulldown the cell lysate
3. Cleave with Ulp1
4. His-pulldown to seperate uncleaved proteins and non-target proteins.
Results
The cleavage of SUMO is done at 4°C for 16 hours. Comparing lanes 3 and 4, it is observed that the cleavage of SUMO with Ulp1 is more than 95% completed. The cleavage products are subjected to a second His pulldown and the flow through is collected (not bound fraction). The procedure removed almost all the SUMO band, showcasing around 90% of His-tag unwanted un-cleaved, and SUMO can be removed with second round pulldown easily.
Further purification could be done with FPLC or HPLC. However, this serves as a proof of concept of the Ulp1 SUMO system for rapid cleavage.
Fig. 1. SDS PAGE comparing cleavage of SUMO with Ulp1
Parts that comprise this composite component
Enhance-T7pro-RBS-Start-6xHis-T7tag - Part:BBa_K4241016
SUMO (Yeast with mutation) - Part:BBa_K4241022
highly engineered mutant of red fluorescent protein from Discosoma striata (coral) - Part:BBa_E1010
Double stop codon - Part:BBa_K4241017
double terminator (B0010-B0012) - Part:BBa_B0015