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Identification and Preliminary Characterization of a Novel Single-Stranded DNA Binding Protein of Staphylococcus aureus Phage Phi11 Expressed in Escherichia coli

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Abstract

Bacteriophage Phi11 harbors a gene, gp13, encoding the putative SSB protein (GenBank accession no. NC_004615.1). SSB proteins bind to and protect the single-stranded DNA molecules from nuclease digestion and are essential for the growth and metabolic activities of the organisms encoding them. In this investigation, we have carried out the cloning, recombinant expression, and purification of rGp13 for the first time in Escherichia coli. EMSA data indicated that the purified recombinant Gp13 protein was capable of binding to single-stranded DNA. The protein exhibited maximum binding activity at 32 °C. Furthermore, our bioinformatic analysis has revealed that Gp13 consists of an OB-fold, a characteristic of SSB proteins. However, the arrangement of the OB-fold is unique, being located in the C-terminal domain of Gp13. Despite the importance of SSB proteins in various metabolic processes as well as in various types of PCR, there are no reports on the purification and characterization of SSB proteins from staphylococcal bacteriophages. We expect that the purification and characterization of recombinant Gp13 will help us gain a better insight into its biological activity and make it available in large quantities for molecular biology work.

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Abbreviations

CD:

Conserved Domain

DNA:

Deoxyribonucleic acid

E. coli:

Escherichia coli

EMSA:

Electrophoretic mobility shift assay

IDT:

Integrated DNA Technologies

IMAC:

Immobilized metal affinity chromatography

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

NCBI:

National Center for Biotechnology Information

Ni–NTA:

Nickel Nitrilotriacetic Acid

OB fold:

Oligosaccharide/ oligonucleotide/ oligopeptide binding fold

PCR:

Polymerase Chain Reaction

PMSF:

Phenylmethane sulfonyl fluoride

RT PCR:

Real Time Polymerase Chain Reaction

S. aureus:

Staphylococcus aureus

SDS-PAGE:

Sodium Dodecyl Sulphate–Polyacrylamide Gel Electrophoresis

SSB:

Single-stranded DNA binding protein

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Acknowledgements

I wish to acknowledge the University Grants Commission, New Delhi, for the financial assistance in the form of Junior Research Fellowship (JRF) and Senior Research Fellowship (SRF), which supported the study.

Funding

This work was supported by the financial assistance from the Department of Science and Technology, Govt. of India (Sanction No. CRG/2020/004968).

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Authors and Affiliations

  1. Department of Biological Sciences, BITS Pilani, K. K. Birla, Goa Campus, NH17B, Zuarinagar, , Goa, 403726, India

    Vivek Ratre, Vijay Hemmadi & Malabika Biswas

  2. VISTA Lab, BITS Pilani, K. K. Birla, Goa Campus, Zuarinagar, Goa, India

    Sumit Biswas

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Ratre, V., Hemmadi, V., Biswas, S. et al. Identification and Preliminary Characterization of a Novel Single-Stranded DNA Binding Protein of Staphylococcus aureus Phage Phi11 Expressed in Escherichia coli. Mol Biotechnol 65, 922–933 (2023). https://doi.org/10.1007/s12033-022-00598-7

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