Difference between revisions of "Part:BBa K3117026"

(→‎Characterization and measuring of the composite part)
 
(21 intermediate revisions by 3 users not shown)
Line 2: Line 2:
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K3117026 short</partinfo>
 
<partinfo>BBa_K3117026 short</partinfo>
 +
  
 
The part <partinfo>BBa_K3117026</partinfo> is a fusion protein of an anti-GPA33 single-chain variable fragment (scFv) and SpyCatcher (<partinfo>BBa_K3117016</partinfo>).
 
The part <partinfo>BBa_K3117026</partinfo> is a fusion protein of an anti-GPA33 single-chain variable fragment (scFv) and SpyCatcher (<partinfo>BBa_K3117016</partinfo>).
  
 
===Usage and Biology===
 
===Usage and Biology===
 +
By connecting the variable regions of the heavy and light chain of an anti-GPA33 antibody with a short, flexible GGGGS linker (<partinfo>BBa_K3117028</partinfo>), the scFv retains it's antigen-binding ability and is much smaller than a conventional antibody. The SpyCatcher attached to the scFv belongs to the SpyTag/SpyCatcher system, of which the sequence is derived of the FbaB protein in the bacterium <i>Streptococcus pyogenes</i>. Once it comes into contact with its corresponding other part, the SpyTag (<partinfo>BBa_K3117015</partinfo>), they bind covalently (Hatlem et al., 2019). This allows our part to be used in a modular manner in combination with other molecules carrying the SpyTag. The sequence contains a C-terminal His-Tag (<partinfo>BBa_K3117005</partinfo>) for easy purification and detection. Secretion of the protein is ensured by an Igk leader (<partinfo>BBa_K3117006</partinfo>), which directs the produced protein into the secretory pathway. When the protein passes the membrane, the leader segment is cleaved off.
  
−
 
+
Since the scFv targets GPA33, which is expressed on more than 95% of colon cancers (Rageul et al., 2009), our part is expected to be optimal for specifically directing its fusion partner (e.g. an effector protein) to colon cancer cells.  
−
 
+
−
The sequence contains a C-terminal His-Tag (<partinfo>BBa_K3117005</partinfo>) for easy purification and detection. Secretion of the protein is ensured by an Igk leader (<partinfo>BBa_K3117006</partinfo>), which directs the produced protein into the secretory pathway. When the protein passes the membrane, the leader segment is cleaved off. By connecting the variable regions of the heavy and light chain of an anti-GPA33 antibody with a short, flexible GGGGS linker (<partinfo>BBa_K3117028</partinfo>), the scFv retains its antigen-binding ability and is much smaller than a conventional antibody.
+
−
 
+
−
The SpyCatcher attached to the scFv belongs to the SpyTag/SpyCatcher system, of which the sequence is derived of the FbaB protein in the bacteria Streptococcus pyogenes. Once it comes into contact with its corresponding other part, the SpyTag (<partinfo>BBa_K3117015</partinfo>), they bind covalently (Hatlem, Trunk, Linke, & Leo, 2019). This allows our part to be used in a modular manner in combination with other molecules carrying the SpyTag.
+
−
 
+
−
Since the scFv targets GPA33, which is expressed on more than 95% of colon cancers (Rageul et al., 2009), our part is expected to be optimal for specifically directing its fusion partner (e.g. an effector protein) to colon cancer cells.
+
  
 
<!-- -->
 
<!-- -->
Line 21: Line 17:
 
===Characterization and measuring of the composite part===
 
===Characterization and measuring of the composite part===
  
−
The construct 2a (K2a) was synthesized by IDT. Therefore, 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.
+
This composite part (K2a) was synthesized by IDT. Then, 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.
  
 
Accuracy of the inserted DNA after cloning was confirmed by sequencing. The data is depicted in Fig. 1.
 
Accuracy of the inserted DNA after cloning was confirmed by sequencing. The data is depicted in Fig. 1.
Line 34: Line 30:
 
Furthermore the His-Tag (<partinfo>BBa_K3117005</partinfo>) in K2a allows purification of the protein with HisTrap columns. The western blot after purification is shown in Fig. 3, depicting the remaining presence of the protein after this step.  
 
Furthermore the His-Tag (<partinfo>BBa_K3117005</partinfo>) in K2a allows purification of the protein with HisTrap columns. The western blot after purification is shown in Fig. 3, depicting the remaining presence of the protein after this step.  
  
−
Bild Aufreinigung
+
[[File:T--FAU_Erlangen--Bild_Sequencing_Aufreinigung_Results_Composite_part.png|thumb|center|150px|'''Figure 3''': Western blot of the constructs after the purification via HisTrap]]
−
[[File:T--FAU_Erlangen--Bild_Sequencing_Aufreinigung_Results_Composite_part.png|thumb|200px|'''Figure 3''': Western blot of the constructs after the purification via HisTrap]]
+
−
 
+
  
−
For the SpyTag/SpyCatcher reaction the protein K2a was added to the protein K2b (<partinfo>BBa_K3117030</partinfo>). 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 might be explained by formation of trimeric protein (Schoene, Fierer, Bennett, & Howarth, 2014).  
+
For the SpyTag/SpyCatcher reaction the protein K2a was added to the protein K2b (<partinfo>BBa_K3117030</partinfo>). 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 might be explained by formation of trimeric protein (Schoene, Fierer, Bennett, & Howarth, 2014).
  
−
Bild Tag/Catcher
+
[[File:T--FAU_Erlangen--Bild_Sequencing_Aufreinigung_TagCatcher_Composite_part.png|thumb|center|150px|'''Figure 4''': Western blot of the constructs K2a, K2b and K2a+b (1:1 ratio K2a with K2b) with an anti His-Tag antibody ]]
−
[[File:T--FAU_Erlangen--Bild_Sequencing_Aufreinigung_TagCatcher_Composite_part.png|thumb|200px|'''Figure 4''': Western blot of the constructs K2a, K2b and K2a+b (1:1 ratio K2a with K2b) with an anti His-Tag antibody ]]
+
  
 
===References===
 
===References===

Latest revision as of 23:25, 21 October 2019


scFv against GPA33 with SpyCatcher codon optimized for CHOs


The part BBa_K3117026 is a fusion protein of an anti-GPA33 single-chain variable fragment (scFv) and SpyCatcher (BBa_K3117016).

Usage and Biology

By connecting the variable regions of the heavy and light chain of an anti-GPA33 antibody with a short, flexible GGGGS linker (BBa_K3117028), the scFv retains it's antigen-binding ability and is much smaller than a conventional antibody. The SpyCatcher attached to the scFv belongs to the SpyTag/SpyCatcher system, of which the sequence is derived of the FbaB protein in the bacterium Streptococcus pyogenes. Once it comes into contact with its corresponding other part, the SpyTag (BBa_K3117015), they bind covalently (Hatlem et al., 2019). This allows our part to be used in a modular manner in combination with other molecules carrying the SpyTag. The sequence contains a C-terminal His-Tag (BBa_K3117005) for easy purification and detection. Secretion of the protein is ensured by an Igk leader (BBa_K3117006), which directs the produced protein into the secretory pathway. When the protein passes the membrane, the leader segment is cleaved off.

Since the scFv targets GPA33, which is expressed on more than 95% of colon cancers (Rageul et al., 2009), our part is expected to be optimal for specifically directing its fusion partner (e.g. an effector protein) to colon cancer cells.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Characterization and measuring of the composite part

This composite part (K2a) was synthesized by IDT. Then, 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.

Accuracy of the inserted DNA after cloning was confirmed by sequencing. The data is depicted in Fig. 1.

Figure 1: Sequencing data of K2a

Fig.2 shows the western blot of the harvest after transfection into HEK 293T cells. For detection, the His-Tag in the K2a sequence (BBa_K3117005) provides the opportunity to be used as a target for a primary antibody in a western blot. K2a can be seen at expected height (42 kDa). Blurry bands might resulted from protease degradation. The presence of K2a in the medium proves the function of the Igk leader (BBa_K3117006), which is directing the protein into the secretory pathway.

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 K2a allows purification of the protein with HisTrap columns. The western blot after purification is shown in Fig. 3, depicting the remaining presence of the protein after this step.

Figure 3: Western blot of the constructs after the purification via HisTrap

For the SpyTag/SpyCatcher reaction the protein K2a was added to the protein K2b (BBa_K3117030). 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 might be explained by formation of trimeric protein (Schoene, Fierer, Bennett, & Howarth, 2014).

Figure 4: Western blot of the constructs K2a, K2b and K2a+b (1:1 ratio K2a with K2b) with an anti His-Tag antibody

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.