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PBP Isolation and DD-Carboxypeptidase Assay

Part of the Methods in Molecular Biology book series (MIMB,volume 1946)

Abstract

Penicillin-binding proteins (PBPs) share the namesake because of their ability to bind penicillin or any beta-lactam antibiotic. In other words, PBPs are the targets of β-lactam antibiotics that hold nearly 60% of the global antibiotic market. These enzymes catalyze the final stages of peptidoglycan (PG) biosynthesis by acting as transglycosylases and transpeptidases. PBPs are also involved in PG remodeling by catalyzing DD-carboxypeptidase (DD-CPase) and endopeptidase reactions. Though the cross-linking abilities of PBPs are well known, the process of remodeling is still unclear, thereby drawing attention toward the DD-CPase enzymes. Here, we describe the step-by-step procedures for isolation of the bacterial cell membrane and detection of PBPs in it, followed by the purification of PBPs (DD-CPases) by both ampicillin-affinity and nickel-nitrilotriacetic acid (Ni-NTA) chromatography. The protocols to determine the enzymatic efficiency are also elucidated. The assays are aimed to determine the kinetic parameters for the interaction of the PBP with BOCILLIN, to evaluate its acylation and deacylation rates, and with its peptide substrates, to assess its DD-CPase activity.

Key words

  • Penicillin-binding protein (PBP)
  • Ampicillin-affinity chromatography
  • Ni-NTA chromatography
  • BOCILLIN
  • Enzyme kinetics
  • Acylation
  • Deacylation
  • DD-Carboxypeptidase assay

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  • DOI: 10.1007/978-1-4939-9118-1_20
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Acknowledgment

The authors are thankful to the past and present members of the Molecular Microbiology Laboratory, Department of Biotechnology, IIT Kharagpur, with special thanks to Mr. Gaurav Kumar, Dr. Satya Deo Pandey, Dr. Chiranjit Chowdhury, Dr. Mouparna Dutta, and Dr. Debasish Kar. SP thanks IIT Kharagpur for her graduate fellowship.

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Correspondence to Anindya S. Ghosh .

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Pal, S., Ghosh, A.S. (2019). PBP Isolation and DD-Carboxypeptidase Assay. In: Biswas, I., Rather, P. (eds) Acinetobacter baumannii. Methods in Molecular Biology, vol 1946. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9118-1_20

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  • DOI: https://doi.org/10.1007/978-1-4939-9118-1_20

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9117-4

  • Online ISBN: 978-1-4939-9118-1

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