Difference between revisions of "Part:BBa K3117026"

Revision as of 19:37, 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

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. 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 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 (BBa_K3117015), 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.

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

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.

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.

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.

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

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

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.

@@ Line 34: / Line 34: @@
 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).
-[[File:T--FAU_Erlangen--Bild_Sequencing_Ernte_Results_Composite_part.png|thumb|center|200px|'''Figure 2''': Western blot of the harvest after transfection into HEK 293T cells with an anti His-Tag antibody]]
+[[File:T--FAU_Erlangen--Bild_Sequencing_Ernte_Results_Composite_part.png|thumb|left|200px|'''Figure 2''': Western blot of the harvest after transfection into HEK 293T cells with an anti His-Tag antibody]]
 [[File:T--FAU_Erlangen--Bild_Sequencing_Aufreinigung_Results_Composite_part.png|thumb|center|200px|'''Figure 3''': Western blot of the constructs after the purification via HisTrap]]
-[[File:T--FAU_Erlangen--Bild_Sequencing_Aufreinigung_TagCatcher_Composite_part.png|thumb|center|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 ]]
+[[File:T--FAU_Erlangen--Bild_Sequencing_Aufreinigung_TagCatcher_Composite_part.png|thumb|right|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===