Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding proteins SMBP and CusF3H+

Jessica J. Gomez-Lugo, Bryan D. Santos, David A. Perez-Perez, Jorge M. Montfort-Gardeazabal, Megan M. McEvoy, Xristo Zarate

Research output: Chapter in Book/Report/Conference proceedingChapter

2 Scopus citations

Abstract

The bacterium Escherichia coli is still considered the first option as a microbial cell factory for recombinant protein production, and affinity chromatography is by far the preferred technique for initial purification after protein expression and cell lysis. In this chapter, we describe the methodology to express and purify recombinant proteins in E. coli tagged with the first two metal-binding proteins proposed as fusion partners. They are the small metal-binding protein SmbP and a mutant of the copper resistance protein CusF3H+. There are several advantages of using them as protein tags: they prevent the formation of inclusion bodies by increasing solubility of the target proteins, they enable purification by immobilized metal-affinity chromatography using Ni(ii) ions with high purity, and because of their low molecular weights, excellent final yields are obtained for the target proteins after cleavage and removal of the protein tag. Here we also describe the protocol for the production of proteins in the periplasm of E. coli tagged with two SmbP variants that include the PelB or the TorA signal sequences for transport via the Sec or the Tat pathway, respectively. Based on these methods, we consider CusF3H+ and SmbP excellent alternatives as fusion proteins for the production of recombinant proteins in E. coli.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages329-344
Number of pages16
DOIs
StatePublished - 2021
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume2178
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Affinity tag
  • CusF3H+
  • Escherichia coli
  • Fusion protein
  • IMAC
  • Metal-affinity chromatography
  • Protein expression and purification
  • SmbP
  • Solubility tag

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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