Analysis of DNA and RNA Binding Properties of Borrelia burgdorferi Regulatory Proteins

  • J. Seshu
  • Trever C. SmithII
  • Ying-Han Lin
  • S. L. Rajasekhar Karna
  • Christine L. Miller
  • Tricia Van Laar
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1690)

Abstract

Bioinformatic approaches and a large volume of prokaryotic genome sequences have enabled rapid identification of regulatory proteins with features to bind DNA or RNA in a given prokaryote. However, biological relevance of these regulatory proteins requires methods to rapidly purify and determine their binding properties within the physiological context or life style of the organism. Here, we describe the experimental approaches to determine the nucleic acid binding properties of regulatory proteins of Borrelia burgdorferi using Borrelia host-adaptation Re.3gulator (BadR—a DNA binding protein) and Carbon storage regulators A of B. burgdorferi (CsrABb—an RNA binding protein) as examples. Best laboratory practices associated with overexpression/purification of recombinant borrelial proteins, synthesis of target nucleic acid sequences, and electrophoretic mobility assays to assess the protein/nucleic acid interactions are described. The methods described are intended to facilitate empirical assessment of the binding affinity, co-factor requirements, quality of the interacting partners, and readily modifiable assay conditions to assess the binding properties to define known and unknown regulatory properties of nucleic acid binding proteins of B. burgdorferi.

Key words

Borrelia burgdorferi Regulatory proteins Protein-nucleic acid interactions Electrophoretic mobility assays 

Notes

Acknowledgments

This study was partly supported by Public Health Service grant SC1-AI-078559 from the National Institute of Allergy and Infectious Diseases, the Army Research Office of the Department of Defense under Contract No. W911NF-11-1-0136, pre-doctoral fellowships from the South Texas Center for Emerging Infectious Diseases (YHL, TAV, SLRK), and the Center For Excellence in Infection Genomics (TCS, CLM, TAV). TCS is supported by a pre-doctoral fellowship from The Brown Foundation.

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Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • J. Seshu
    • 1
    • 2
  • Trever C. SmithII
    • 1
    • 2
  • Ying-Han Lin
    • 1
    • 2
  • S. L. Rajasekhar Karna
    • 1
    • 2
  • Christine L. Miller
    • 1
    • 2
  • Tricia Van Laar
    • 1
    • 2
  1. 1.Department of BiologyThe University of Texas at San AntonioSan AntonioUSA
  2. 2.South Texas Center for Emerging Infectious Diseases (STCEID), Center of Excellence in Infection Genomics (CEIG)The University of Texas at San AntonioSan AntonioUSA

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