Part:BBa_K2382010

Anti-aflatoxin scFv fusion protein construction DNA sequence

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1342
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 403
  Illegal SapI.rc site found at 316
  

Usage and Biology

By ligating the constitutive promoter (BBa_J23101), strong ribosome binding site (BBa_B0034) and scFv-EAAAK- RFP-Histag, we were able to detect aflatoxin by the purified protein expressing in E. coli. Moreover, we designed a restriction site, BamHI, between scFv and EAAAK, so future iGEM teams could take advantage of this composite part to fuse their scFv with RFP as indicator, and make their own test strip!

Characterization of the Anti-antitoxin scFv

Expression results

Initially when we were designing the sequence of scFv, our plan was to replace the coloring function of gold nanoparticle with RFP(BBa_K2382010). But the colonies we got after the first transformation did not present visible red colors as we expected. We were not sure whether the problem came from RFP itself, or the function of RFP being affected by other proteins, causing it to fail to show desired results. After the fusion protein purification, we obtained a 57kDa single band that is the same size as our protein, so we were sure that the fusion protein has been produced (Fig.10).

Fig.10: This result show that bands in 57 kDa were the purified fusion protein. Flow：flow through, Wash：protein washed by wash buffer, E1~E5：Elution1~Elution5

Later we analyzed the binding capacity of the purified protein. In the experiment we first went through dialysis of excess salt and then diluted the samples 2000 and 4000 times before doing ELISA assay. After our test, only 2000 times dilution of the protein seems to perform a bit of affinity (Fig. 11).

Fig.11: ELISA assay test 2000 times dilution of the scFv fusion protein. If antibody have binding ability, the O.D value would change with AFB1 level. Horizontal axis means Aflatoxin B1 concentration. Vertical axis means value of O.D 450 nm.

ELISA

However, we doubted that the result might be interrupted by salt in buffer. Therefore, we narrowed the dilution range to 2000 times to 4000 times (Fig. 12), and desalted before ELISA assay. If antibody have binding ability, the O.D value would change with AFB1 level. Theoretically, the OD we measured would increase with the concentration of aflatoxin. However, the trend line did not follow this rule; the antibody may not perform any binding ability. Unfortunately from the final results we can only judge that the fusion protein does not have any binding ability.

After subsequent discussion with the instructor, we suspect that the binding ability of scFv is fine, but after the addition of other proteins, the original binding ability may have been change, which led to the loss of the original function.

Fig.12: ELISA assay test 4000 times dilution of the ScFv fusion protein. In this time we desalted and increased the protein concentration to reduce interference. However, the result does not match with assumption. Horizontal axis means Aflatoxin B1 concentration. Vertical axis means value of O.D 450 nm.

References

(1)Morrison SL. Cloning, expression, and modification of antibody V regions. Curr Protoc Immunol. 2002 May;Chapter 2:Unit 2.12. doi: 10.1002/0471142735.im0212s47.

(2)Biing-Hui Liu, KuangeChun Chu, Feng-Yih Yu. Novel monoclonal antibody-based sensitive enzyme-linked immunosorbent assay and rapid immunochromatographic strip for detecting aflatoxin M1 in milk. Food Control 66 (2016) 1-7

(3)Reddy Chichili, V. P., Kumar, V., & Sivaraman, J. (2013). Linkers in the structural biology of protein–protein interactions. Protein Science : A Publication of the Protein Society, 22(2), 153–167. http://doi.org/10.1002/pro.2206

(4): X. Chen, et al., Fusion protein linkers: Property, design and functionality, Adv. Drug Deliv. Rev. (2012), http:// dx.doi.org/10.1016/j.addr.2012.09.039

(5)Xin Li, Peiwu Li, Qi Zhang, Yuanyuan Li, Wen Zhang & Xiaoxia Ding. Molecular Characterization of Monoclonal Antibodies against Aflatoxins: A Possible Explanation for the Highest Sensitivity. © 2012 American Chemical Society dx.doi.org/10.1021/ac202747u | Anal. Chem. 2012, 84, 5229−5235