Composite

Part:BBa_K2885002

Designed by: Sungho Ko   Group: iGEM18_BioMarvel   (2018-10-04)


Gold binding polypeptide (GBP) + Protein G (ProG) Fusion

In our study, gold binding polypeptide (GBP)-Protein G (ProG)(GBP-ProG), BBa_K2885002, was constructed from Integrated DNA Technologies (IDT) and the sequence was analyzed by BIOFACT™ sequencing analysis service. After the construct was inserted into pSB1C3 vector, the recombinant vector’s transformation and amplification were perfromed in E. coli DH5-alpha competent cell with IPTG-induced expression. With the TALON metal affinity resin, the GBP-ProG fusion protein was highly purified. The purified protein was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and then quantified using the Bradford protein assay to dilute to 1 mg/ml with a phosphate buffered solution (PBS) for experimental use in this study. Figure 1 shows the result of SDS-PAGE to verify exact size scale of the GBP-ProG.



To shed light on successful immobilization of antibody by the GBP-ProG on the gold surface, we applied the GBP-ProG to the gold surface in the microchannel of our chip, followed by injection of the alkaline phosphatase (AP)-labeled immunoglobulin G (IgG) to immobilize onto the GBP-ProG formed. AP-labeled IgG also was loaded on a bare gold surface inside the microchannel as a control. Then PAPP was treated into both microchannels to observe cyclic voltammograms (CV) induced by its enzymatic reaction as a substrate. Figure 2 shows that CV current values were 38.63 μA at conditions in which GBP-ProG exists in spite of no cyclic voltammetric peak responses in without GBP-ProG. This result showed that GBP-ProG was successfully immobilized onto the gold surface via the GBP portion, indicating the oriented binding of antibodies onto the ProG domain targeting the Fc region of antibodies. Similarly, Park et al. (2014) reported that GBP-ProG, encoded in pET-GBP-ProG plasmid, showed rapid and simple binding onto a gold surface and immobilization of antibodies, compared to other chemical methods, which suggests that GBP-ProG can be an alternative to the chemical crosslinkers used in the present. Hence, our GBP-ProG can be an efficient crosslinker of diverse antibodies.



References

[1] Park, J.H., Kim, Y.P., Kim, I.H., Ko, S. Rapid detection of aflatoxin B1 by a bifunctional protein crosslinker-based surface plasmon resonance biosensor. (2014). Food Control 36(1), 183-190.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 644


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