Part:BBa_K4241023

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.

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