Difference between revisions of "Part:BBa K4241023"

 
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__NOTOC__
 
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<partinfo>BBa_K4241023 short</partinfo>
 
<partinfo>BBa_K4241023 short</partinfo>
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<partinfo>BBa_K4241023 SequenceAndFeatures</partinfo>
  
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This is the expression of a T7 tagged SUMO-RFP fusion, which the RFP could be his-tag pulled down and purified via Ulp1 cleavage and second round of His-tag pulldown.
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===Overview===
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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.
  
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<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
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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.
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<br/>
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Purification scheme:
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1. Express the fusion protein in E. coli and sonicate cells
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2. His-pulldown the cell lysate
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3. Cleave with Ulp1
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4. His-pulldown to seperate uncleaved proteins and non-target proteins.
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===Results===
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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
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K4241023 SequenceAndFeatures</partinfo>
 
  
  

Revision as of 16:16, 9 October 2022


6xHis_SUMO_RFP


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 155
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 301
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1050
    Illegal AgeI site found at 1162
  • 1000
    COMPATIBLE 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.

BBa K4241023-1.jpg

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