Part:BBa_K3829004

V5-Tag V5 tag is a short peptide tag for detection and purification of proteins. The V5 tag can be fused/cloned to a recombinant protein and detected in ELISA, flow cytometry, immunoprecipitation, immunofluorescence, and Western blotting with antibodies and Nanobodies.

Improvement: IvyMaker-China 2022 iGEM Team

Characterization- [Contribution] Change the position of V5 Tag

What we have learned and want to share with iGEMers: From this session, we understand that in protein expression/surface display systems, protein folding problems need to be considered in particular. And sometimes protein folding problems can be solved by changing the position of tags or proteins. V5 tag is a basic part used last year (BBa_K3829004), this year we introduced Tag-catcher system. When replacing RFP and GFP with MHETase and PETase, we did not observe immunofluorescence with secondary antibodies that should theoretically bind specifically to the V5 tag. To analyze whether PETase-spytag and MHETase-snooptag fused protein folded correctly, we constructed a model of the fusion protein, we used prediction software such as trRosetta and ITASSER to construct the structure. The evaluation results of the two models shows the structure is convincing. So, no enzyme activity could be a steric hindrance between the fusion protein and the scaffold (See Modeling for details, model link here).

Similarly, we used I-TASSER to model our “CBM-SC-SC-SNC-SC-V5-7813” scaffold. When the display system is constructed, immunofluorescence cannot be detected, presumably as the V5 tag has been obstructed. To verify the theory, we predicted the model of the overall protein using the I-TASSER server and discovered that the V5 tag is truly embedded by other proteins.

Fig.1 Model of scaffold CBM-SC-SC-SNC-SC-V5-7813 predicted by I-TASSER server.

It can be seen from the figure that the red component (V5 tag) is blocked by other components, meaning the V5 tag cannot function ideally as designed. We presumed the V5 tag would be available if it was located at the sequence's beginning, as the catchers may have a larger size that blocks the V5 tag if it is located at the end of the sequence.

Fig.2 Model of scaffold CBM-V5-SC-SC-SNC-SC-7813 predicted by I-TASSER server.

To make V5 tag and in turn immunofluorescence visible, we changed V5 tag’s position to the front of the plasmid. This edition means V5 tag transcription takes place before catchers’ transcription, lowering the possibility that large seized catcher protein obstructing V5-tag. After altering the V5 tag’s location, we predicted the model again using I-TASSER to ensure its feasibility.

Finally, the results of changing the position of V5 tag were proved to be effective.

Fig.3 Successful construction of changing the position of V5-tag.

References

[1] Wei Zheng, Chengxin Zhang, Yang Li, Robin Pearce, Eric W. Bell, Yang Zhang. Folding non-homology proteins by coupling deep-learning contact maps with I-TASSER assembly simulations. Cell Reports Methods, 1: 100014 (2021).

[2] Chengxin Zhang, Peter L. Freddolino, and Yang Zhang. COFACTOR: improved protein function prediction by combining structure, sequence and protein-protein interaction information. Nucleic Acids Research, 45: W291-299 (2017).

[3] Jianyi Yang, Yang Zhang. I-TASSER server: new development for protein structure and function predictions, Nucleic Acids Research, 43: W174-W181, 2015.

[4] Lu, Hongyuan, et al. "Machine learning-aided engineering of hydrolases for PET depolymerization." Nature 604.7907 (2022): 662-667.

Sequence and Features BBa_K3829004 SequenceAndFeatures