Investigation of the Effect of Arginine and Glutathione on Recovery of a Single Domain Antibody Produced in Bacteria in Inclusion Bodies

Abstract

Single domain antibodies (nanobodies) are the antigen binding domain of heavy chain only antibodies derived from camelids and sharks. Standard antibodies have heavy and light chains, and the antigen binding region is formed by the combination of variable heavy and variable light chain domains. Single domain antibodies are as good binders to their respective antigens as the combination of variable heavy and light chains. Due to their chemical and thermal stability, small size, and economic benefits, there is increasing interest in nanobodies for research use and from industry. In this article, we investigated the effect of arginine and a mixture of oxidized and reduced glutathione for recovering a nanobody produced in inclusion bodies in E. coli. Nanobody protein is solubilized in 6M urea buffer from the cell lysate. After a Ni-NTA chromatography, nanobody containing fractions were first diluted in different concentrations of arginine and/or reduced and oxidized glutathione containing buffers, followed by dialysis against a buffer to fold the protein. The best recovery yield was obtained in the presence of 400mM arginine. Nanobodies are important molecules in biotechnology and medicine, and, this study investigated ways to improve their production yield.

Keywords

• Protein recovery
• Single domain antibody
• Arginine
• Glutathione

References

1. Arakawa, T., & Ejima, D. (2014). Refolding technologies for antibody fragments. Antibodies, 3(3), 232–241.
2. Arakawa, T., Ejima, D., Tsumoto, K., Obeyama, N., Tanaka, Y., Kita, Y., & Timasheff, S. N. (2007). Suppression of protein interactions by arginine: A proposed mechanism of the arginine effects. Biophysical Chemistry, 127(1-2), 1–8.
3. Ban, B., Sharma, M., & Shetty, J. (2020). Optimization of methods for the production and refolding of biologically active disulfide bond-rich antibody fragments in microbial hosts. Antibodies, 9(3), 39–57.
4. Bao, X., Xu, L., Lu, X., & Jia, L. (2016). Optimization of dilution refolding conditions for a camelid single domain antibody against human beta-2-microglobulin. Protein Expression and Purification, 117, 59–66.
5. Berkmen, M. (2012). Production of disulfide-bonded proteins in Escherichia coli. Protein Expression and Purification, 82(1), 240–251.
6. Bhatwa, A., Wang, W., Hassan, Y. I., Abraham, N., Li, X.-Z., & Zhou, T. (2021). Challenges associated with the formation of recombinant protein inclusion bodies in Escherichia coli and strategies to address them for industrial applications. Frontiers in Bioengineering and Biotechnology, 9, 630551.
7. Birnboim-Perach, R., Grinberg, Y., Vaks, L., Nahary, L., & Benhar, I. (2019). Production of stabilized antibody fragments in the E. coli bacterial cytoplasm and in transiently transfected mammalian cells. In Human Monoclonal Antibodies: Methods and Protocols (Vol. 1904, pp. 455–480). Springer.
8. Bocedi, A., Cattani, G., Gambardella, G., Ticconi, S., Cozzolino, F., Di Fusco, O., Pucci, P., & Ricci, G. (2019). Ultra-rapid glutathionylation of ribonuclease: Is this the real incipit of its oxidative folding? International Journal of Molecular Sciences, 20(21), 5440.

9. Dingus, J. G., Tang, J. C. Y., Amamoto, R., Wallick, G. K., & Cepko, C. L. (2022). A general approach for stabilizing nanobodies for intracellular expression. eLife, 11, e68253.
10. Gezehagn, K. G., & Tessema, T. S. (2024). The potential of single-chain variable fragment antibody: Role in future therapeutic and diagnostic biologics. Journal of Immunology Research, 2024(1), 1804038.
11. Hamers-Casterman, C., Atarhouch, T., Muyledermans, S., Robinson, G., Hamers, C., Bajyana Songa, E., Bendarham, N., & Hamers, R. (1993). Naturally occurring antibodies devoid of light chains. Nature, 363(6428), 446–448.
12. Helma, J., Schmidthals, K., Lux, V., Nüske, S., Scholz, A. M., Kräusslich, H. G., Rothbauer, U., & Leonhardt, H. (2012). Direct and dynamic detection of HIV-1 in living cells. PLoS One, 7(11), e50026. https://doi.org/10.1371/journal.pone.0050026
13. Hennigan, J. N., Menacho-Melgar, R., Sarkar, P., Golovsky, M., & Lynch, M. D. (2024). Scalable, robust, high-throughput expression & purification of nanobodies enabled by 2-stage dynamic control. Metabolic Engineering, 85, 116–130.
14. Maggi, M., & Scotti, C. (2017). Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies. Protein Expression and Purification, 136, 39–44.
15. Manta, B., Boyd, D., & Berkmen, M. (2019). Disulfide bond formation in the periplasm of Escherichia coli. EcoSal Plus, 8(2). https://doi.org/10.1128/ecosalplus.ESP-0010-2018
16. Martins, A. C., Oshiro, Y. M., Schiavon, B. N., de Jesus, G. A., de la Torre, B. G., & Albericio, F. (2025). Monoclonal antibodies (mAbs) and proteins: The biologic drugs approved by the FDA in 2024. Biomedicines, 13(8), 1962.
17. Muyldermans, S. (2021). Applications of nanobodies. Annual Review of Animal Biosciences, 9(1), 401–421.
18. Okumura, M., Saiki, M., Yamaguchi, H., & Hidaka, Y. (2011). Acceleration of disulfide-coupled protein folding using glutathione derivatives. FEBS Journal, 278(7), 1137–1144.
19. Rees, A. (2021). Antibodies: A history of their discovery and properties. In F. Rüker & G. Wozniak-Knopp (Eds.), Introduction to Antibody Engineering (1st ed., pp. 5–40). Springer Nature.
20. Rudolph, R., & Lilie, H. (1996). In vitro folding of inclusion body proteins. The FASEB Journal, 10(1), 49–56.
21. The Antibody Society. (2025). Therapeutic monoclonal antibodies approved or in review in the EU or US. https://www.antibodysociety.org/resources/approved-antibodies.
22. Yamaguchi, S., Yamamoto, E., Mannen, T., & Nagamune, T. (2013). Protein refolding using chemical refolding additives. Biotechnology Journal, 8(1), 17–31.
23. Yasuda, M., Murakami, Y., Sowa, A., Ogino, H., & Ishikawa, H. (1998). Effect of additives on refolding of a denatured protein. Biotechnology Progress, 14(4), 601–606.