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Cell-free expression, purification and immunoreactivity assessment of recombinant Fasciola hepatica saposin-like protein-2

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Abstract

Cell free protein synthesis has become a powerful method for the high-throughput production of proteins that are difficult to express in living cells. The protein SAP2 of Fasciola hepatica (FhSAP2), which has demonstrated to be both, an excellent vaccine candidate against experimental fascioliasis and a good antigen for serodiagnosis of human chronic fascioliasis, is a typical example of a molecule that is difficult to produce. This is mainly due to its tendency to get over-expressed in inclusion bodies by prokaryotes. FhSAP2 expressed in an Escherichia coli-based expression system is poorly glycosylated, insoluble and often undergoes improper folding leading it to reduced immunogenicity. In this work, FhSAP2 was expressed in vitro using the eukaryote cell free system, TNT T7 Quick coupled transcription/translation, that has been designed for the expression of PCR-generated DNA templates. FhSAP2 was expressed in micro-volumes and purified by an affinity chromatography method, which gave a protein yield of 500 µg/ml as determined by bicinchoninic acid assay method. Circular dichroism, Western blotting and enzyme-linked immunosorbent assay analysis were used to confirm the secondary structure, purity and integrity of protein. Results demonstrate that FhSAP2 can be expressed in a cell-free system retaining its main conformational and antigenic properties. The protein purified could be used in immunization experiments and immunodiagnostic techniques.

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Acknowledgements

This research was supported by Grant Numbers G12MD007600, R25GM061838 from the National Institute on Minority Health and Health Disparities and from Puerto Rico Louis Stokes Alliance for Minority Participation-Bridge to the Doctorate Program Fellowship; Funded by the National Sciences Foundation, NSF Grant Award HRD-1400870 and Support Competitive Research (SCORE) Research Advancement (SC1) Grant Award 1SC1AI096108-01A2. The content is solely responsibility of the authors and does not necessary represent the official views of NSF.

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Author notes

  1. Marcos J. Ramos-Benítez and Luis M. Lopez-Cruz have contributed equally.

Authors and Affiliations

  1. Department of Microbiology, University of Puerto Rico, Medical Sciences Campus, PO Box 365067, San Juan, PR, 00936-5067, USA

    Marcos J. Ramos-Benítez, Vasti Aguayo, Caleb Ruiz-Jimenez & Ana M. Espino

  2. Department of Biology, Faculty of Natural Sciences, University of Puerto Rico, Rio Piedras Campus, San Juan, PR, 00931, USA

    Luis M. Lopez-Cruz

Authors
  1. Marcos J. Ramos-Benítez
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  2. Luis M. Lopez-Cruz
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  3. Vasti Aguayo
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  4. Caleb Ruiz-Jimenez
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  5. Ana M. Espino
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Contributions

Conceived and designed the experiments: MJR-B, LML-C, AME. Performed the experiments: MJR-B, LML-C, VA, CR-J. Analyzed data: MJR-B, LML-C, AME. Provided resources and laboratory space: AME. Wrote the paper: AME. Made the Proof-reading: VA.

Corresponding author

Correspondence to Ana M. Espino.

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The authors of the current manuscript declare that there is no potential conflict of interest.

Research involving animal rights

Rabbits used in the study were maintained in the facilities of the Animal Resources Center of Medical Sciences Campus, University of Puerto Rico, and all experiments were performed under an MSC-IACUC-approved animal study protocol (No. 7870110), which follows AAALAC guidelines.

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Ramos-Benítez, M.J., Lopez-Cruz, L.M., Aguayo, V. et al. Cell-free expression, purification and immunoreactivity assessment of recombinant Fasciola hepatica saposin-like protein-2. Mol Biol Rep 45, 1551–1556 (2018). https://doi.org/10.1007/s11033-018-4251-3

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  • DOI: https://doi.org/10.1007/s11033-018-4251-3

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