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Prokaryotic Expression, In Vitro Biological Analysis, and In Silico Structural Evaluation of Guinea Pig IL-4

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Abstract

Interleukin-4 is a signature cytokine of T-helper type 2 (Th2) cells that play a major role in shaping immune responses. Its role in highly relevant animal model of tuberculosis (TB) like guinea pig has not been studied till date. In the current study, the guinea pig IL-4 gene was cloned and expressed using a prokaryotic expression vector (pET30 a(+)). This approach yielded a recombinant protein of 19 kDa as confirmed by mass spectrometry analysis and named as recombinant guinea pig (rgp)IL-4 protein. The authenticity of the expression of rgpIL-4 protein was further verified through polyclonal anti-IL4 antiserum raised in rabbits that showed specific and strong binding with the recombinant protein. The biological activity of the rgpIL-4 was ascertained in RAW264.7 cells where LPS-treated nitric oxide (NO) production was found to be suppressed in the presence of this protein. The three-dimensional structure of guinea pig IL-4 was predicted by utilizing the template structure of human interleukin-4, which shared a sequence homology of 58%. The homology modeling result showed clear resemblance of guinea pig IL-4 structure with the human IL-4. Taken together, our study indicates that the newly expressed, biologically active rgpIL-4 protein could provide deeper understanding of the immune responses in guinea pig to different infectious diseases like TB and non-infectious ones.

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Acknowledgements

The authors thank Vignan’s University for providing facilities under FIST to execute this work. The authors also thank NIT, Rourkela for providing facilities to execute some of the experiments. The authors acknowledge the support of SERB (ECR/2016/00304). We also thank the anonymous reviewers for their comments which helped us in improving the quality of the manuscript.

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Author notes

  1. Madhavan Omanakuttan, Hanumohan R. Konatham, and Vijaya R. Dirisala are co-authors who contributed equally to this study.

Authors and Affiliations

  1. Department of Biotechnology, Vignan’s Foundation for Science, Technology & Research, Guntur, Andhra Pradesh, 522213, India

    Madhavan Omanakuttan, Hanumohan R. Konatham & Vijaya R. Dirisala

  2. Department of Microbial Pathogenesis and Immunology, College of Medicine, Texas A&M Health Science Center, College Station, TX, 77843, USA

    Amminikutty Jeevan, Lan H. Ly & David McMurray

  3. Laboratory of Mycobacterial Immunology, Department of Life Science, National Institute of Technology, Rourkela, Odisha, 769008, India

    Shradha Mawatwal & Rohan Dhiman

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  1. Madhavan Omanakuttan
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Correspondence to Vijaya R. Dirisala.

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Supplementary material 1 (PPTX 344 kb)

Supplementary figure 1. Structural validation of SAVES server. Figure (a) shows the Ramachandran plot for the predicted structure. (b) PROVE server showing the atomic resolution and (c) ERRAT2 score showing the overall quality factor. Supplementary figure 2. RAW264.7 cells were seeded overnight and stimulated next day with either LPS (10 µg/ml) or left alone for 48 h. Some of the combinations were pretreated with either rgpIL-4 protein or rgpIL-4 protein generated using codon-optimized IL-4 gene for 1 h before adding LPS. The nitrite assay was performed in harvested supernatants using Griess reagent as described in Materials and Methods. The data shown represent values of 3 independent experiments and are represented as Mean ± SEM. ** denotes p < 0.01 and * denotes p < 0.05.

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Omanakuttan, M., Konatham, H.R., Dirisala, V.R. et al. Prokaryotic Expression, In Vitro Biological Analysis, and In Silico Structural Evaluation of Guinea Pig IL-4. Mol Biotechnol 62, 104–110 (2020). https://doi.org/10.1007/s12033-019-00227-w

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