PKS–NRPS Enzymology and Structural Biology: Considerations in Protein Production

Meredith A. Skiba, Finn P. Maloney, Qingyun Dan, Amy E. Fraley, Courtney C. Aldrich, Janet L. Smith, W. Clay Brown

Research output: Chapter in Book/Report/Conference proceedingChapter

6 Scopus citations

Abstract

The structural diversity and complexity of marine natural products have made them a rich and productive source of new bioactive molecules for drug development. The identification of these new compounds has led to extensive study of the protein constituents of the biosynthetic pathways from the producing microbes. Essential processes in the dissection of biosynthesis have been the elucidation of catalytic functions and the determination of 3D structures for enzymes of the polyketide synthases and nonribosomal peptide synthetases that carry out individual reactions. The size and complexity of these proteins present numerous difficulties in the process of going from gene to structure. Here, we review the problems that may be encountered at the various steps of this process and discuss some of the solutions devised in our and other labs for the cloning, production, purification, and structure solution of complex proteins using Escherichia coli as a heterologous host.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
EditorsBradley S. Moore
PublisherAcademic Press Inc.
Pages45-88
Number of pages44
ISBN (Print)9780128139592
DOIs
StatePublished - Jan 1 2018

Publication series

NameMethods in Enzymology
Volume604
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Bibliographical note

Funding Information:
This work has been supported by the NIH (R01-DK42303, R01-GM076477, R01-CA108874 to J.L.S. and R01-AI070219 to C.C.A.) and by the Margaret J. Hunter Professorship to J.L.S. M.A.S. has been supported by predoctoral fellowships from an NIH Cellular Biotechnology Training Program (T32-GM008353) and the University of Michigan Rackham Graduate School.

Keywords

  • Chaperones
  • Coexpression
  • Cyanobacteria
  • Nonribosomal peptide synthetase
  • Polyketide synthase
  • Protein crystallization
  • Protein folding
  • Protein production
  • Protein purification
  • Protein solubility

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