Part:BBa_K4197008
OmpA_Ara h 2 fusion
Gene fusion to express the peanut allergen Ara h 2 on the surface of E. coli.
Introduction
This part is composed of the gene coding for the allergen of peanut Ara h 2 (NCBI: AY158467.1). The peanut allergy prevalence is higher than 5% (Lieberman and al. 2018) in developped countries and Ara h 2, among the 17 other peanut allergens, triggers 90% of peanut allergies (Lehmann and al. 2003). Ara h 2 have already been expressed in E. coli and was able to bind the IgE of patient with peanut's allergie (Lehmann and al. 2003). Ara h 2 was merged to the membrane protein OmpA of E. coli (BBa_K1694002) to display Ara h on the surface of E. coli. This lipoprotein is the most abundant in E. coli membrane with 100,000 copies per cell (Ortiz-Suarez and al. 2016) and is often used to display protein on the surface of bacteria (Yang and al. 2016).
Construction
Ara h 2 gene ordered on IDT was amplified by PCR using the high fidelity Phusion polymerase with primers IF3_allergen F (gccgcaagctttaatgatggtgatggtgatggtgatg) and IF4_Ara h 2 (cctgtattttcagagcatggccaagctcaccattc). Expected size of the amplicon was 567 bp.
To merge Ara h 2 to OmpA, the gene was inserted in a pET-21 b (+) plasmid containing OmpA merged to Gal d 2, another allergen. The plasmid was linearized, excluding the Gal d 2 fragment by PCR. The primers used were IF1_allergen and IF2_plasmid. Expected size of the amplicon was 5924 bp.
Amplification product sizes were checked on Ethidium bromide stained agarose gel (Figure 6).
Amplification products matched expected sizes, they were further purified from the gel.
The Ara h 2 fragment was then inserted into pET-21 b (+)_OmpA by In-Fusion. The In-Fusion assemby reaction was transformed into Stellar competent cells. Transformants were selected on LB-ampicillin plates. 15 transformants were then screened by colony PCR with primer pairs flanking the insertion zone (primers used: screening_inserts-F: ggttatgctagttattgctcagc and screening_inserts-R: ccgaaacaagcgctcatgagc, expected size of the amplicons: 1450 bp). 13 positive transformants were detected (Figure 7).
Four of these transformants (colonies 21, 22, 23, 24) had their plasmid extracted by Miniprep. Sequences were all validated by Sanger sequencing. The plasmid was named pET-21 b (+)_OmpA_Ara h 2.
The plasmid was finally used to transform E. coli Tuner cells to express OmpA_Ara h 2 at the cell membrane.
Expression controls
We assessed the expression of the allergens at the outer surface of the bacteria, an important control experiment prior to aggregation experiments. Two experiments were conducted to do so: one with a fluorescent anti-HIS-tag IgG and one with an IgE coupled to a fluorescent anti-IgE IgG.
Test of Ara h 2 protein expression by HIS-tag detection
As the protein was HIS-tagged, we first tried to detect it by binding a fluorescent anti-HIS-tag IgG to the cell membrane and measuring fluorescence. Cells without IgG were observed as negative control. Microscope observations are shown in Figure 16. The microscope parameters to observe GFP were the following: excitation filter 360/40, dichroic beamsplitter 400 nm, emission filter 470/40.
No difference in fluorescence was visible between the control and the sample. The only fluorescence observed was due to autofluorescence of cells, so we could not conclude if the protein was expressed at the membrane, especially without a positive control (like binding the protein to a HIS-tag resin and observing fluorescence).
Test of the allergens functionality by double antibody recognition (ELISA test)
We assessed the functionality of the allergens by binding specific IgE to them (anti-Ara h 2 and anti-Der p 1), and then binding a fluorescent anti-IgE IgG to the complex. No fluorescence was observed in either case (data not shown). Without a positive control, we could not conclude between an incorrect exposition of the protein (not facing the outer surface), or a mere problem during the experimental setup.
Conclusion
In conclusion, validation remains to be performed to assess this part functionality.
References
More information about the project for which the part was created: DAISY (INSA-UPS 2022)
Other parts to display allergens:
- OmpA_Gal d 2
- OmpA_Ana o 3
- OmpA_Der p 1
- Lieberman, J., Sublett, J., Ali, Y., Haselkorn, T., Damle, V., Chidambaram, A., Rosen, K., & Mahr, T. (2018). INCREASED INCIDENCE AND PREVALENCE OF PEANUT ALLERGY IN CHILDREN AND ADOLESCENTS IN THE UNITED STATES. Annals of Allergy, Asthma & ; Immunology, 121(5), S13. https://doi.org/10.1016/j.anai.2018.09.039
- Lehmann, K., Hoffmann, S., Neudecker, P., Suhr, M., Becker, W.-M., & Rösch, P. (2003). High-yield expression in Escherichia coli, purification, and characterization of properly folded major peanut allergen Ara h 2. Protein Expression and Purification, 31(2), 250–259. https://doi.org/10.1016/s1046-5928(03)00190-6
- Ortiz-Suarez, M. L., Samsudin, F., Piggot, T. J., Bond, P. J., & Khalid, S. (2016). Full-Length OmpA : Structure, Function, and Membrane Interactions Predicted by Molecular Dynamics Simulations. Biophysical Journal, 111(8), 1692–1702. https://doi.org/10.1016/j.bpj.2016.09.009
- Yang, Chao; Zhao, Qiao; Liu, Zheng; Li, Qiyun; Qiao, Chuanling; Mulchandani, Ashok; et al. (2016): Cell Surface Display of Functional Macromolecule Fusions on Escherichia coli for Development of an Autofluorescent Whole-Cell Biocatalyst. ACS Publications. Journal contribution. https://doi.org/10.1021/es800441t.s001
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 130
Illegal XbaI site found at 47 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 130
Illegal NheI site found at 92
Illegal NotI site found at 1188 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 130
Illegal BamHI site found at 124
Illegal BamHI site found at 813
Illegal BamHI site found at 834
Illegal XhoI site found at 1197 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 130
Illegal XbaI site found at 47 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 130
Illegal XbaI site found at 47 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 713
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