Difference between revisions of "Part:BBa K4241026"
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double terminator (B0010-B0012) - Part:BBa_B0015 <br/> | double terminator (B0010-B0012) - Part:BBa_B0015 <br/> | ||
6xHis_SUMO_CeB - Part:BBa_K4241024 <br/> | 6xHis_SUMO_CeB - Part:BBa_K4241024 <br/> | ||
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| + | ===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. | 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. | ||
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Latest revision as of 07:38, 10 October 2022
6xHis_Ulp1
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 155
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Overview
This composite part is for 6xHis-tagged Ulp1. This is to be purified via his-tag pulldown. Further purification is possible with FPLC and size exclusion chromatography or ion exchange chromatograpgy.
Usage and Biology
This is a bacterial expression system for 6xHis-Ulp1 fusion protein. By expressing the T7 tagged 6xHis-Ulp1 fusion protein via an enhanced T7pCONS system, we are able to increase the expression yield of the protein.
Ulp1 is an enzyme designed to cleave the SUMO-tag from it's fusion protein. This reaction is specific allowing for the specific and induced cleavage of SUMO. Further, Ulp1 can be purified via his-tag pulldown. Further purification is possible with FPLC and size exclusion chromatography or ion exchange chromatograpgy.
This is the coding sequence for 6xHis-Ulp1 to be used in bacterial protein expression systems.
Parts that comprise this composite component
Enhance-T7pro-RBS-Start-6xHis-T7tag - Part:BBa_K4241016
Ulp1 N-terminal (SUMO protease) - Part:BBa_K4241025
Double stop codon - Part:BBa_K4241017
double terminator (B0010-B0012) - Part:BBa_B0015
6xHis_SUMO_CeB - Part:BBa_K4241024
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