Difference between revisions of "Part:BBa K4241023"
DaLegends6 (Talk | contribs) |
DaLegends6 (Talk | contribs) |
||
| Line 11: | Line 11: | ||
<br/> | <br/> | ||
Purification scheme: | Purification scheme: | ||
| − | 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. | ||
Revision as of 16:23, 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