Difference between revisions of "Part:BBa K4241026"

 
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K4241026 short</partinfo>
 
<partinfo>BBa_K4241026 short</partinfo>
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<partinfo>BBa_K4241026 SequenceAndFeatures</partinfo>
  
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This is a 6xHis-tagged Ulp1 expression. It can be purified via his-tag pulldown of beads. Then further purification is possible with FPLC and size exclusion or ion exchange chromatography.
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===Overview===
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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.
  
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<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===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. <br/>
 +
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. <br/>
 +
This is the coding sequence for 6xHis-Ulp1 to be used in bacterial protein expression systems.
  
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<!-- -->
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===Parts that comprise this composite component===
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<span class='h3bb'>Sequence and Features</span>
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Enhance-T7pro-RBS-Start-6xHis-T7tag - Part:BBa_K4241016 <br/>
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<partinfo>BBa_K4241026 SequenceAndFeatures</partinfo>
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Ulp1 N-terminal (SUMO protease) - Part:BBa_K4241025 <br/>
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Double stop codon - Part:BBa_K4241017 <br/>
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double terminator (B0010-B0012) - Part:BBa_B0015 <br/>
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6xHis_SUMO_CeB - Part:BBa_K4241024 <br/>
 +
 
 +
===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.
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<br/>
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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.
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[[File:BBa K4241023-1.jpg|center]]
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Fig. 1. SDS PAGE comparing cleavage of SUMO with Ulp1
  
  

Latest revision as of 07:38, 10 October 2022


6xHis_Ulp1


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 155
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE 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.

BBa K4241023-1.jpg

Fig. 1. SDS PAGE comparing cleavage of SUMO with Ulp1