Difference between revisions of "Part:BBa K3117030"
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The construct 2b (K2b) was synthesized by IDT. It was cloned into the expression vector pSEC/tag2/Hygro\C\-\OK by Gibson Assembly. Our protein was under control of the CMV promotor ensuring a high-level constitutive expression. Afterwards, the construct was brought into HEK293T cells with either calcium phosphate transfection or lipofection.
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Construct 2b (K2b) was synthesized by IDT. It was cloned into the expression vector pSEC/tag2/Hygro\C\-\OK by Gibson Assembly. Our protein was under control of the CMV promotor ensuring a high-level constitutive expression. Afterwards, the construct was brought into HEK293T cells with either calcium phosphate transfection or lipofection.
  
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The accuracy of the inserted DNA after cloning was confirmed with sequencing. The data is depicted in Fig. 1.
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To assure DNA was correctly inserted during cloning samples were sequenced. Data is depicted in Fig. 1.
  
  
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Figure 1: Sequencing data of the complete K2b
 
Figure 1: Sequencing data of the complete K2b
  
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Fig. 2 shows the harvest after transfection into HEK 293T cells in a western blot. For detection, the His-Tag in the K2b sequence (BBa_K3117005) provides the opportunity to be used as a target for a primary antibody in a western blot.  
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Fig. 2 shows the harvest after transfection of HEK 293T cells in a western blot. For detection, the His-Tag in the K2b sequence (BBa_K3117005) provides the opportunity to be used as a target for a primary antibody in a western blot.  
 
K2b can be seen at expected height (41 kDa). Presence of K2b in the medium proves the function of the Igk leader (BBa_K3117006), which is directing the protein into the secretory pathway.  
 
K2b can be seen at expected height (41 kDa). Presence of K2b in the medium proves the function of the Igk leader (BBa_K3117006), which is directing the protein into the secretory pathway.  
  
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Bild TagCatcher
 
Bild TagCatcher
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Figure 4: Western blot after SpyTag/SpyCatcher reaction  
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Figure 4: Western blot after SpyTag/SpyCatcher reaction
  
 
===References===
 
===References===

Revision as of 19:03, 21 October 2019


scFv against CD3 with SpyTag codon optimized for CHOs

BBa_K3117026 is a fusion protein of an anti-CD3 single-chain variable fragment (scFv) and SpyTag (BBa_K3117015).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 604
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Characterization and measurement

Construct 2b (K2b) was synthesized by IDT. It was cloned into the expression vector pSEC/tag2/Hygro\C\-\OK by Gibson Assembly. Our protein was under control of the CMV promotor ensuring a high-level constitutive expression. Afterwards, the construct was brought into HEK293T cells with either calcium phosphate transfection or lipofection.

To assure DNA was correctly inserted during cloning samples were sequenced. Data is depicted in Fig. 1.


Bild Sequencing K2b Figure 1: Sequencing data of the complete K2b

Fig. 2 shows the harvest after transfection of HEK 293T cells in a western blot. For detection, the His-Tag in the K2b sequence (BBa_K3117005) provides the opportunity to be used as a target for a primary antibody in a western blot. K2b can be seen at expected height (41 kDa). Presence of K2b in the medium proves the function of the Igk leader (BBa_K3117006), which is directing the protein into the secretory pathway.

Bild Ernte Figure 2: Western blot of the harvest after transfection into HEK 293T cells with an anti-His-Tag antibody

Furthermore the His-Tag (BBa_K3117005) in K2b allows purification of the protein with HisTrap columns. The western blot after this purification is shown in Fig. 3, showing the remaining presence of the protein after this step.

Bild Aufreinigung Figure 3: Western blot of the purified protein with HisTrap columns with an anti-His_tag antibody

For the SpyTag/SpyCatcher reaction the protein K2b was added to the protein K2a (BBa_K3117026). A height of 83 kDa was expected in the afterwards performed western blot, depicted in Fig 4. A specific band appeared after adding the two products, but this band was running at ~120 kDa. This size could be explained by formation of trimeric protein (Schoene, Fierer, Bennett, & Howarth, 2014).

Bild TagCatcher Figure 4: Western blot after SpyTag/SpyCatcher reaction

References

1. Hatlem, D., Trunk, T., Linke, D., & Leo, J. C. (2019). Catching a SPY: Using the SpyCatcher-SpyTag and Related Systems for Labeling and Localizing Bacterial Proteins. International journal of molecular sciences, 20(9), 2129.

2. Rageul, J., Mottier, S., Jarry, A., Shah, Y., Théoleyre, S., Masson, D., . . . Denis, M. G. (2009). KLF4‐dependent, PPARγ‐induced expression of GPA33 in colon cancer cell lines. International journal of cancer, 125(12), 2802-2809.

3. Schoene, C., Fierer, J. O., Bennett, S. P., & Howarth, M. (2014). SpyTag/SpyCatcher cyclization confers resilience to boiling on a mesophilic enzyme. Angewandte Chemie International Edition, 53(24), 6101-6104.