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Extraction and Characterizations of Viral Protein Particles: A Methodological Study

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Abstract

Phage (viral) tailspike proteins (TSP) are used in many translation applications such as in pathogenic bacteria detection and therapeutics. Especially, bacterial virus (or bacteriophage) P22 TSP is one of the most popular viral proteins in molecular virology and is very well-studied in terms of structure, interactions with O-antigen (a component of bacterial cell wall), and various aspects of its functions using X-ray crystallography and in vitro and in vivo studies. Although there are many papers on the virological and structural aspects of this protein, basic lab-based methodological studies and its solution-based characterizations using an in-house affordable experimental method which can be easily followed in a laboratory at an undergraduate or graduate level are missing in the literature. Our idea here is to provide a detailed method for preparing P22 TSP in a laboratory environment and studying its basic characterizations in the solution state using the available methods in our laboratory. These methods include SDS-PAGE and Native-PAGE for molecular weight determination, as well as specificity and affinity testing against its host bacterium. Our studies using SDS-PAGE show a band for TSP in the monomeric state at around 72 kDa, and the Native-PAGE proves the trimeric state with a size of around 250 kDa, thereby confirming the information regarding molar mass. On the other side, UV-Vis spectroscopy verifies absorption properties that lie around 250–300 nm. Furthermore, the solution structures of proteins in the native state, determined using dynamic light scattering (DLS), exhibit various size distributions around 10 nm, 100 nm, and 1000 nm, indicating the coexistence of trimeric and multimeric states, along with a small number of aggregates. Finally, the affinity of the TSP with the O-antigen of the host bacterium has been confirmed through western blotting studies, and the results are discussed in the context of the role of different intermolecular interactions involved in the binding of the TSP and the O-antigen group using molecular docking approach. We strongly believe our basic studies will be very helpful for beginners and graduate students in the related field.

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Acknowledgements

PSM acknowledges BIRAC, Center of Excellence project, Department of Biotechnology, Govt. Of India , Depart of Biotechnology and ICMR (Indian Council of Medical Research), Government of India, for support in the current work.

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  1. Manoswini Manoswini and Madhuchhanda Mohanty are joint first author.

Authors and Affiliations

  1. School of Biotechnology, Kalinga Institute of Industrial Technology (KIIT), Deemed to be University, Bhubaneswar, Odisha, India

    Manoswini Manoswini, Madhuchhanda Mohanty, Amrito Ghosh Majumdar, Bikash R. Sahu & Priti Sundar Mohanty

  2. Department of Zoology, School of Applied Science, Centurion University of Technology and Management, Jatni, Odisha, India

    Bikash R. Sahu

  3. School of Chemical Technology, Kalinga Institute of Industrial Technology (KIIT), Deemed to be University, Bhubaneswar, Odisha, India

    Priti Sundar Mohanty

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Contributions

PSM: principal investigator of the work, conceptualization of the problem, formulation of paper, methodology, carried out some parts of experiments, critical analysis and interpretation of all experiments.

Manoswini: experimental work, synthesized the proteins, characterized and prepared the original draft in the protein study.

MM: theoretical understanding and in silico molecular docking simulation study

AGM: referencing, correction, editing,

BRS: supervision and useful suggestion in the discussion.

The entire manuscript has undergone a comprehensive revision by MM. AGM assisted in referencing and some editing work in the revised manuscript. This revision included the correction of English grammar throughout the manuscript, restructuring of sentences and paragraphs, detailed docking methodology, and studies. Additionally, figures were revised to enhance resolution, and references were updated

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Correspondence to Priti Sundar Mohanty.

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Manoswini, M., Mohanty, M., Majumdar, A.G. et al. Extraction and Characterizations of Viral Protein Particles: A Methodological Study. BioNanoSci. (2024). https://doi.org/10.1007/s12668-023-01289-6

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