Difference between revisions of "Part:BBa K4365021"
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===Usage and Biology===
 
===Usage and Biology===
  
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====Cloning strategy====
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====Proof of His-tag functionality====
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[[File:BBa_K4365021_histag.png|300px|thumb|Figure 12: SDS-PAGE gel of the pellet of SP-SUMO-turboRFP expressing yeast cells, before and after batch binding (BB). The lysed pellet, the supernatant of the culture, the lysed pellet after batch binding, and the supernatant after batch binding were examined. Protein Ladder= Thermo Scientific PageRuler Plus Prestained Protein Ladder.]]
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After verifying that the construct was secreted into the growth medium, we performed batch binding purification on a 500 ml yeast culture grown in MV medium for 3 days. The pellet obtained after centrifugation and the supernatant were used for [batch-binding purification](https://www.notion.so/1bac1a97cb4343c1ad46760e40e2489f).
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Four samples were obtained from the purification procedure: lysed pellet, supernatant,  lysed pellet after batch binding, and supernatant after batch binding. These samples were used to run [an SDS/Page gel and a Western blot](https://www.notion.so/Protein-Analysis-0d67ad0091fd4f28918ebe033534c611) using antibodies against turboRFP and the His6 tag.
 +
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The Coomassie stain (Figure 12) showed that the supernatant contained fewer secreted proteins compared to the pellet and indicated that indeed a secreted protein would already possess a high degree of purity relative to a protein extracted by cell lysis.
 +
 +
The Western blot revealed that the full SP-SUMO turboRFP construct was present in the transformed yeast cells (Figure 13). Two protein bands were visible on the lane corresponding to the lysed pellet of cells. One band at ~25 kDa corresponds to the turboRFP size. The band at ~60 kDa, we hypothesize, corresponds to the SP-SUMO-turboRFP (~25 kDa + 13 kDa) bound to the Ulp1 protease (~29 kDa). This indicated that a good portion of the protein was already cleaved before leaving the cell. Sill the supernatant contained secreted turboRFP without SP-SUMO (Figure 13). The batch binding purification was able to capture the SP-SUMO from the lysed pellet but not from the supernatant.
 +
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[[File:BBa_K4365021_wb.png|700px|center|thumb|Figure 13: Western blot with anti-turboRFP (A) and anti-His6 antibodies (B). The lysed pellet, the supernatant of the culture, the lysed pellet after batch binding (BB), and the supernatant after batch binding (BB) were analyzed. Protein Ladder= Thermo Scientific PageRuler Plus Prestained Protein Ladder.]]
  
  

Revision as of 08:31, 11 October 2022


SP-SUMO-turboRFP

SP-SUMO-tRFP is a reporter device which could be used to test protein secretion in yeast. Moreover, if tRFP is exchanged for the protein of interest, after protein purification with His-tag you can scarlessly remove the tag from the protein with a SUMO protease. This part is optimized for expression in Saccharomyces cerevisiae.

SP-SUMO-tRFP consists of:


Figure 1: illustration of SP-SUMO system for secretion and scar-less protein production in yeast. The SP-SUMO is attached to the cargo protein turboRFP as this was the construct we tested during our experiments. Created with Biorender.com.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 1001
    Illegal PstI site found at 1036
    Illegal PstI site found at 1335
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 1001
    Illegal PstI site found at 1036
    Illegal PstI site found at 1335
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2168
    Illegal BamHI site found at 1007
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 1001
    Illegal PstI site found at 1036
    Illegal PstI site found at 1335
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 1001
    Illegal PstI site found at 1036
    Illegal PstI site found at 1335
    Illegal NgoMIV site found at 172
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

Cloning strategy

Proof of His-tag functionality

Figure 12: SDS-PAGE gel of the pellet of SP-SUMO-turboRFP expressing yeast cells, before and after batch binding (BB). The lysed pellet, the supernatant of the culture, the lysed pellet after batch binding, and the supernatant after batch binding were examined. Protein Ladder= Thermo Scientific PageRuler Plus Prestained Protein Ladder.

After verifying that the construct was secreted into the growth medium, we performed batch binding purification on a 500 ml yeast culture grown in MV medium for 3 days. The pellet obtained after centrifugation and the supernatant were used for [batch-binding purification](https://www.notion.so/1bac1a97cb4343c1ad46760e40e2489f).

Four samples were obtained from the purification procedure: lysed pellet, supernatant, lysed pellet after batch binding, and supernatant after batch binding. These samples were used to run [an SDS/Page gel and a Western blot](https://www.notion.so/Protein-Analysis-0d67ad0091fd4f28918ebe033534c611) using antibodies against turboRFP and the His6 tag.

The Coomassie stain (Figure 12) showed that the supernatant contained fewer secreted proteins compared to the pellet and indicated that indeed a secreted protein would already possess a high degree of purity relative to a protein extracted by cell lysis.

The Western blot revealed that the full SP-SUMO turboRFP construct was present in the transformed yeast cells (Figure 13). Two protein bands were visible on the lane corresponding to the lysed pellet of cells. One band at ~25 kDa corresponds to the turboRFP size. The band at ~60 kDa, we hypothesize, corresponds to the SP-SUMO-turboRFP (~25 kDa + 13 kDa) bound to the Ulp1 protease (~29 kDa). This indicated that a good portion of the protein was already cleaved before leaving the cell. Sill the supernatant contained secreted turboRFP without SP-SUMO (Figure 13). The batch binding purification was able to capture the SP-SUMO from the lysed pellet but not from the supernatant.

Figure 13: Western blot with anti-turboRFP (A) and anti-His6 antibodies (B). The lysed pellet, the supernatant of the culture, the lysed pellet after batch binding (BB), and the supernatant after batch binding (BB) were analyzed. Protein Ladder= Thermo Scientific PageRuler Plus Prestained Protein Ladder.