Heterologous expression, purification, and characterization of proteins in the lanthanome

Emily R. Featherston, Joseph A. Mattocks, Jonathan L. Tirsch, Joseph A. Cotruvo

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Scopus citations

Abstract

Recent work has revealed that certain lanthanides—in particular, the more earth-abundant, lighter lanthanides—play essential roles in pyrroloquinoline quinone (PQQ) dependent alcohol dehydrogenases from methylotrophic and non-methylotrophic bacteria. More recently, efforts of several laboratories have begun to identify the molecular players (the lanthanome) involved in selective uptake, recognition, and utilization of lanthanides within the cell. In this chapter, we present protocols for the heterologous expression in Escherichia coli, purification, and characterization of many of the currently known proteins that comprise the lanthanome of the model facultative methylotroph, Methylorubrum extorquens AM1. In addition to the methanol dehydrogenase XoxF, these proteins include the associated c-type cytochrome, XoxG, and solute binding protein, XoxJ. We also present new, streamlined protocols for purification of the highly selective lanthanide-binding protein, lanmodulin, and a solute binding protein for PQQ, PqqT. Finally, we discuss simple, spectroscopic methods for determining lanthanide- and PQQ-binding stoichiometry of proteins. We envision that these protocols will be useful to investigators identifying and characterizing novel members of the lanthanome in many organisms.

Original languageEnglish (US)
Title of host publicationRare-Earth Element Biochemistry
Subtitle of host publicationMethanol Dehydrogenases and Lanthanide Biology
EditorsJoseph A. Cotruvo
PublisherAcademic Press Inc.
Pages119-157
Number of pages39
ISBN (Print)9780128238561
DOIs
StatePublished - Jan 2021

Publication series

NameMethods in Enzymology
Volume650
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Medicine(all)

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