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Bacterial production and biophysical characterization of a hard-to-fold scFv against myeloid leukemia cell surface marker, IL-1RAP

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Abstract

Background

Interleukin-1 receptor accessory protein (IL-1RAP) is one of the most promising therapeutic targets proposed for myeloid leukemia. Antibodies (Abs) specific to IL-1RAP could be valuable tools for targeted therapy of this lethal malignancy. This study is about the preparation of a difficult-to-produce single-chain variable fragment (scFv) construct against the membrane-bound isoform of human IL-1RAP using Escherichia coli (E. coli).

Methods

Different approaches were examined for refolding and characterization of the scFv. Binding activities of antibody fragments were comparatively evaluated using cell-based enzyme-linked immunosorbent assay (ELISA). Homogeneity and secondary structure of selected scFv preparation were analyzed using analytical size exclusion chromatography (SEC) and circular dichroism (CD) spectroscopy, respectively. The activity of the selected preparation was evaluated after long-term storage, repeated freeze-thaw cycles, or following incubation with normal and leukemic serum.

Results

Strategies for soluble expression of the scFv failed. Even with the help of Trx, ≥ 98% of proteins were expressed as inclusion bodies (IBs). Among three different refolding methods, the highest recovery rate was obtained from the dilution method (11.2%). Trx-tag substantially enhanced the expression level (18%, considering the molecular weight (MW) differences), recovery rate (˃1.6-fold), and binding activity (˃2.6-fold increase in absorbance450nm). The produced scFv exhibited expected secondary structure as well as acceptable bio-functionality, homogeneity, and stability.

Conclusion

We were able to produce  21 mg/L culture functional and stable anti-IL-1RAP scFv via recovering IBs by pulse dilution procedure. The produced scFv as a useful targeting agent could be used in scheming new therapeutics or diagnostics for myeloid malignancies.

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Acknowledgements

The authors wish to express their deep appreciation to all personnel of “Venom and Biotherapeutic Molecules Laboratory” and “Protein Chemistry Laboratory” of Pasteur Institute of Iran. The authors also particularly thank Dr. Kamran Pooshang Bagheri, Dr. Vahid Khalaj, Dr. Saman Rahmati, Dr. Zahra Mohammadi, Mahsa Hashemi, Shadi Damough, Saeedeh Pouri, Dr. Rada Dehghan, and Zahra Khodayari for their sincere help.

Funding

This study was extracted from the Ph.D. thesis of the first author and financially supported by the Pasteur Institute of Iran (grant number: BD-9365). This work was also partly supported by Iran National Science Foundation (INSF) (grants number 96011491).

Author information

Authors and Affiliations

  1. Medical Biotechnology Department, Biotechnology research center, Pasteur Institute of Iran (IPI), No. 69, Pasteur Ave, Tehran, Iran

    Aref Farokhi-Fard, Elham Bayat, Arezoo Beig Parikhani, Samira Komijani, Farzaneh Barkhordari & Fatemeh Davami

  2. Student Research Committee, Pasteur Institute of Iran, Tehran, Iran

    Aref Farokhi-Fard

  3. Protein Chemistry Laboratory, Medical Biotechnology Department, Biotechnology research center, Pasteur Institute of Iran, Tehran, Iran

    Hooman Aghamirza Moghim Aliabadi

  4. Advance Chemical Studies Laboratory, Faculty of Chemistry, K.N. Toosi University, Tehran, Iran

    Hooman Aghamirza Moghim Aliabadi

  5. Drug Design and Bioinformatics Unit, Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran

    Soroush Sardari

  6. Oncology Department, Naft Hospital, Tehran, Iran

    Behrouz Gharib

  7. Departement of Virology, Pasteur Institute of Iran, Tehran, Iran

    Kayhan Azadmanesh

  8. Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran

    Morteza Karimipoor

  9. Department of Nanobiotechnology, New Technology Research Group, Pasteur Institute of Iran, Tehran, Iran

    Haleh Bakhshandeh

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  1. Aref Farokhi-Fard
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Corresponding author

Correspondence to Fatemeh Davami.

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Ethical statement

The authors are responsible for the correctness of the statements provided in the manuscript. All procedures performed in studies involving human participants were in accordance with the 1964 Helsinki Declaration and its later amendments and with the ethical standards of the Pasteur Institute of Iran’s research ethics committee (code of ethics: IR.PII.REC.1401.032). Written informed consent was obtained from the patient volunteer. This article does not contain any studies with animals.

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Farokhi-Fard, A., Bayat, E., Beig Parikhani, A. et al. Bacterial production and biophysical characterization of a hard-to-fold scFv against myeloid leukemia cell surface marker, IL-1RAP. Mol Biol Rep 50, 1191–1202 (2023). https://doi.org/10.1007/s11033-022-07972-3

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