Difference between revisions of "Part:BBa K2933132"

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===Usage and Biology===
 
===Usage and Biology===
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This composite part is made up with four basic parts, the His tag, the Sumo tag, three cutting sites(NdeI, NheI and BamHI) and our target protein SHD. It encodes a protein which is SHD fused with His-Sumo tag. The fusion protein is about 41.0kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of SHD and combine the three parts with these three cutting sites. The fusion protein can be cut off at the cutting site BamHI. It is convenient for us to purify our target protein.<br>
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This composite part is made up with five basic parts, the His tag, the Sumo tag, three cutting sites(NdeI, NheI and BamHI) and our target protein SHD. It encodes a protein which is SHD fused with His-Sumo tag. The fusion protein is about 41.0kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of SHD and combine the three parts with these three cutting sites. The fusion protein can be cut off at the cutting site BamHI. It is convenient for us to purify our target protein.<br>
  
 
===Molecular cloning===
 
===Molecular cloning===
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First, we used the vector pGEX-6p-1 to construct our expression plasmid. And then we converted the plasmid constructed to ''E. coli'' DH5α to expand the plasmid largely.<br>
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First, we used the vector pET28b-sumo to construct our expression plasmid. And then we converted the plasmid constructed to ''E. coli'' DH5α to expand the plasmid largely.<br>
 
<p style="text-align: center;">
 
<p style="text-align: center;">
 
   [[File:SHD-PCR.png|500px]]<br>
 
   [[File:SHD-PCR.png|500px]]<br>
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After verification, it was determined that the construction is successful. We converted the plasmid to ''E. coli'' BL21(DE3) for expression and purification.<br>
 
After verification, it was determined that the construction is successful. We converted the plasmid to ''E. coli'' BL21(DE3) for expression and purification.<br>
  
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===Expression and purification===
 
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'''Pre-expression:'''<br>
 
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The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.<br>
 
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'''Massive expressing:'''<br>
 
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After taking samples, we transfered them into 1L LB medium and add antibiotic to 100 μg/mL final concentration. Grow them up in 37°C shaking incubator. Grow until an OD 600 nm of 0.8 to 1.2 (roughly 3-4 hours). Induce the culture to express protein by adding 1 mM IPTG (isopropylthiogalactoside, MW 238 g/mol). Put the liter flasks in 16°C shaking incubator for 16h.<br>
 
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'''Affinity Chromatography:'''<br>
 
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We used the GST Agarose to purify the target protein. The GST Agarose can combine specifically with the GST tag fused with target protein. <br>
 
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* First, wash the column with GST-binding buffer for 10 minutes to balance the GST column.<br>
 
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* Second, add the protein solution to the column, let it flow naturally and bind to the column.<br>
 
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* Third, add GST-Washing buffer several times and let it flow. Take 10μl of wash solution and test with Coomassie Brilliant Blue. Stop washing when it doesn’t turn blue.<br>
 
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* Forth, add 400μL Prescission Protease (1mg/mL) to the agarose. Digest for 16 hours in 4℃.
 
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* Fifth, add GST-Elution buffer several times. Check as above. Collect the eluted proteins for further operation.<br>
 
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<p style="text-align: center;">
 
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    [[File:T--TJUSLS China--SHD GST.jpg|400px]]<br>
 
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'''Figure 2.'''  The result of SDS-page.<br>
 
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</p>
 
  
 
==References==
 
==References==

Latest revision as of 11:38, 25 September 2019


His+Linker a+Sumo+Linker b+SHD

This part encodes the fusion protein of His tag, sumo tag and SHD to promote the expression and purification of target protein(SHD).


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 256
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 256
    Illegal NheI site found at 33
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 256
    Illegal BglII site found at 145
    Illegal BamHI site found at 344
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 256
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 256
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This composite part is made up with five basic parts, the His tag, the Sumo tag, three cutting sites(NdeI, NheI and BamHI) and our target protein SHD. It encodes a protein which is SHD fused with His-Sumo tag. The fusion protein is about 41.0kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of SHD and combine the three parts with these three cutting sites. The fusion protein can be cut off at the cutting site BamHI. It is convenient for us to purify our target protein.

Molecular cloning

First, we used the vector pET28b-sumo to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.

SHD-PCR.png
Figure 1. Left: The PCR result of SHD. Right: The verification results by enzyme digestion.

After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.


References