Small ubiquitin-like modifier-tag and modified protein purification significantly increase the quality and quantity of recombinant African swine fever virus p30 protein

Vet World Vol.17 May-2024 Article - 25

Research Article

Veterinary World, 17(5): 1157-1167

https://doi.org/10.14202/vetworld.2024.1157-1167

Jullada Chootip¹, Payuda Hansoongnern^1,2, Nattarat Thangthamniyom¹, Sirin Theerawatanasirikul³, Penpitcha Chankeeree¹, Challika Kaewborisuth², and Porntippa Lekcharoensuk^1,4

1. Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, 10900, Thailand.
2. Virology and Cell Technology Research Team, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathumthani, 12120, Thailand.
3. Department of Anatomy, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, 10900, Thailand.
4. Center for Advanced Studies in Agriculture and Food, KU Institute of Advanced Studies, Kasetsart University, Bangkok, 10900, Thailand.

Background and Aim: African swine fever (ASF) is a highly virulent and contagious viral disease caused by the ASF virus (ASFV). It has a significant impact on swine production throughout the world, while existing vaccines and specific treatments remain ineffective. ASFV p30 is a potent antigenic protein that induces protective antibodies immediately after infection; however, most recombinant p30 is insoluble. This study aimed to improve the solubility, yield, and purity of recombinant p30 by tagging it with a small ubiquitin-like modifier (SUMO) and modifying the protein purification process.

Materials and Methods: SUMO fused with ASFV p30 (SUMO-p30) and p30 alone were cloned and expressed in Escherichia coli. SUMO-p30 and p30 solubility and expression levels were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein purification was modified by combining ammonium sulfate precipitation method with affinity chromatography. In addition, large-scale production of all versions of p30 were compared using SDS-PAGE and western blotting, and the purified p30 was used to develop the indirect enzyme-linked immunosorbent assay (ELISA).

Results: The solubility and expression levels of SUMO-p30 were dramatically enhanced compared with that of p30. Modification of the purification process significantly increased purified and soluble SUMO-p30 and p30 yields by 6.59 and 1.02 μg/mL, respectively. Large-scale production confirmed that this procedure increased the quantity of recombinant p30 while maintaining protein purity and immunogenicity. The p30-based indirect ELISA was able to discriminate between positive and negative serum samples with statistically significant differences in mean optical density 450 values (p < 0.001).

Conclusion: This study demonstrates the enhancement of solubility, purity, and yield of ASFV p30 expressed in E.coli by SUMO fusion tagging and combining ammonium sulfate precipitation with affinity chromatography for protein purification. These positive effects were sustained in large-scale production. Cleavage and removal of hexahistidine-SUMO tag from the fusion protein by protease may not be suitable when handling a large amount of the protein. However, the SUMO-fused p30 retained strong immunoreactivity to convalescent swine serum, indicating its application in immunization and diagnostic purposes. The expression and purification procedures in this study could be applied to increase solubility, quality, and quantity of other recombinant proteins as well.

Keywords: African swine fever, diagnosis, indirect enzyme-linked immunosorbent assay, p30, protein expression, purification.

How to cite this article: Chootip J, Hansoongnern P, Thangthamniyom N, Theerawatanasirikul S, Chankeeree P, Kaewborisuth C, and Lekcharoensuk P (2024) Small ubiquitin-like modifier-tag and modified protein purification significantly increase the quality and quantity of recombinant African swine fever virus p30 protein, Veterinary World, 17(5): 1157-1167.

Received: 2024-01-28 Accepted: 2024-04-30 Published online: 2024-05-22

Corresponding author: Porntippa Lekcharoensuk E-mail: fvetptn@ku.ac.th

DOI: 10.14202/vetworld.2024.1157-1167

Copyright: Chootip, et al. This article is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/ by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.