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 ). Similarly, we used I-TASSER to model our “CBM-SC-SC-SNC-SC-V5-7813” scaffold (See Modeling for details). 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 The chemical structure of FAST-PETase.
We also measured the effectiveness of FAST-PETase more directly by testing its effect with degrading PET powder. Specifically, we took the following steps. First, we collected an appropriate amount of cultivated strains and washed it three times with 50 mM glycine-NaOH (pH 9.0-10) buffer. Second, the bacteria were incubated with 1 mL buffer containing 50 mM glycine-NaOH (pH 9.0) and 10 mg PET powder at 30℃ with a speed of 900 r/min. Third, the reaction was terminated by diluting the aqueous solution with 18 mM phosphate buffer (pH 2.5) containing 10% (v/v) DMSO followed by heat treatment (85°C, 10 min). Fourth, the supernatant obtained by centrifugation (15,000 × g, 10 min) was analyzed by HPLC. The result shown in the figure below reflected a significantly larger concentration of degraded PET and MHET with FAST-PETase than wild PETase, consistent under different OD conditions.
Fig.2 Comparison of enzyme activities of fast and wild PETase.
Fig.3 Comparison of FAST-PETase and wild PETase with HPLC analysis of degraded PET.
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| None |